Biological fertilizer compositions comprising sludge

ABSTRACT

The present invention provides biological fertilizer compositions that comprise yeast cells and sludge. The yeast cells of the invention have an enhanced ability to fix atmospheric nitrogen, decompose phosphorus minerals and compounds, decompose potassium minerals and compounds, decompose complex carbon compounds, overproduce growth factors, overproduce ATP, decompose undesirable chemicals, suppress growth of pathogenic microorganisms, or reduce undesirable odor. The biological fertilizer composition of the invention can replace mineral fertilizers in supplying nitrogen, phosphorus, and potassium to crop plants. Methods of manufacturing biological fertilizer compositions, and methods of uses are also encompassed.

1. FIELD OF THE INVENTION

The invention relates to biological fertilizers that comprise yeasts andan organic substrate. The yeasts in the compositions of the inventionhave been stimulated to perform a variety of functions including theconversion of the organic materials into non-hazardous plant nutrients.The invention also relates to methods for manufacturing biologicalfertilizers, and methods for using the biological fertilizers toincrease crop yields.

2. BACKGROUND OF THE INVENTION

Use of fertilizer is essential in supporting the growth of high yieldcrops. Of the basic nutrients that plants need for healthy growth, largeamounts of nitrogen (taken up as NO₃ ⁻ or NH₄ ⁺), phosphorus (taken upas H₂PO₄ ⁻), and potassium (taken up as K⁺) nutrients are required bymost crops on most soils (Wichmann, W., et al., IFA World Fertilizer UseManual). Such large amounts of nitrogen, phosphorus, and potassiumnutrients are supplied mainly in the form of mineral fertilizers, eitherprocessed natural minerals or manufactured chemicals (K. F. Isherwood,1998, Mineral Fertilizer Use and the Environment, United NationsEnvironmental Programme Technical Report No. 26).

Despite the importance of mineral fertilizers in providing mankind withabundant agricultural products, the harm done to the environment hasbeen recognized in recent years. Mineral fertilizers may incurreddamages to soils. For example, most nitrogen fertilizers may acidifysoils, thereby adversely affecting the growth of plants and other soilorganisms. Extensive use of chemical nitrogen fertilizers may alsoinhibit the activity of natural nitrogen fixing microorganisms, therebydecreasing the natural fertility of soils. The long term use of mineralfertilizers may also cause severe environmental pollution. For example,the loss of nitrogen and phosphate fertilizers due to leaching and soilerosion has led to contamination of soil and ground water, andeutrophication of surface water. Cleaning up polluted soil and water hasbeen a complicated and difficult task. The cost for such a task is alsoastronomical.

In search for a solution to the problem, some are going back to organicfertilizers, such as manure (Wichmann, W., et al., IFA World FertilizerUse Manual). The use of manure as fertilizer dates to the beginnings ofagriculture. Large amounts of manure are produced by livestock. Forexample, in the United States, farms (including confined animal feedingoperations) generate more than 136 million metric tons (dry weightbasis) of waste products annually. Manure has value in maintaining andimproving soil because of the plant nutrients, humus, and organicsubstances contained in it. Studies have shown that a high percentage ofthe nitrogen, phosphorus, and potassium fed to dairy cattle are excretedin manure.

As manure must be managed carefully in order to derive the most benefitfrom it, some farmers may be unwilling to expend the necessary time andeffort. Manure must be carefully stored to minimize loss of nutrients.It must be applied to the right kind of crop at the proper time. Ingeneral, manure does not provide all the plant nutrients needed and verylarge amount of organic fertilizers have to be applied to soil. Thus,there is a tendency to discount the value of manure as fertilizer.Manure may also contain undesirable chemicals, such as antibiotics andhormones. Only in underdeveloped countries, where artificial fertilizermay be costly or unavailable and where labor is relatively cheap, manureis attractive as a fertilizer.

Furthermore, manure may contain significant levels of nitrogen andphosphorous which threaten water resources if not managed correctly. Ifnot stored or disposed of properly, it can pose health and environmentalthreats. For example, it can cause air pollution, i.e., odor and dust;and contamination of surface and ground water with excess nutrients,organic matter, salts, and pathogens. For example, manure containspathogenic microorganisms, such as Escherichia coli Salmonella spp.,Shigella spp., and Campylobacter jejuni.

Biological fertilizers utilizing microorganisms have been proposed asalternatives to mineral fertilizers. Naturally occurring nitrogen fixingmicroorganisms including bacteria, such as Rhizobium, Azotobacter, andAzospirillum, (See for example, U.S. Pat. No. 5,071,462) and fungi, suchas Aspergillus flavus-oryzae, (See, for example, U.S. Pat. No.4,670,037) have been utilized in biological fertilizers. Naturallyoccurring microorganisms capable of solubilizing phosphate rock ore orother insoluble phosphates into soluble phosphates have also beenutilized in biological fertilizers either separately (e.g., U.S. Pat.No. 5,912,398) or in combination with nitrogen fixing microorganisms(e.g., U.S. Pat. No. 5,484,464). Genetically modified bacterial strainshave also been developed and utilized in biological fertilizers. Anapproach based on recombinant DNA techniques has been developed tocreate more effective nitrogen fixing, phosphorus decomposing, andpotassium decomposing bacterial strains for use in a biologicalfertilizer, see, for example, U.S. Pat. No. 5,578,486; PCT publicationWO 95/09814; Chinese patent publication: CN 1081662A; CN 1082016A; CN1082017A; CN 1103060A; and CN 1109595A.

However, the biological fertilizers that are based on naturallyoccurring microorganisms are generally not efficient enough toeffectively replace mineral fertilizers. It is therefore important todevelop more advanced biological fertilizers that can replace mineralfertilizers in supplying nitrogen, phosphorus, and potassium to cropsfor producing high quality agricultural products while avoiding theproblems associated with mineral fertilizers.

The present invention provides a biological fertilizer based onnon-recombinant yeasts, which can replace mineral fertilizers andprovide an effective and environmentally-friendly method of usingcertain organic materials.

Citation of documents herein is not intended as an admission that any ofthe documents cited herein is pertinent prior art, or an admission thatthe cited documents are considered material to the patentability of theclaims of the present application. All statements as to the date orrepresentations as to the contents of these documents are based on theinformation available to the applicant and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

3. SUMMARY OF THE INVENTION

The present invention relates to biological fertilizer compositions. Thebiological fertilizer compositions of the invention comprises up to ninedifferent yeast cell components, sludge, and optionally an inorganicsubstrate component. In particular, the yeast cell components of thecomposition are each capable of at least one of the following tenfunctions, namely, fixing atmospheric nitrogen, decomposing insolublephosphorus or potassium minerals, maintaining a balance of phosphoruscompounds, decomposing complex carbon-containing materials or compounds,overproducing growth factors, overproducing ATP, suppression of growthof pathogenic microorganisms, breakdown of undesirable chemicals, andreducing the odor of organic matters, respectively. The yeast cellcomponents of the invention can be used as an additive which is mixedwith sludge to form a biological fertilizer.

In one embodiment, the biological fertilizer compositions of theinvention are produced by mixing sludge with at least seven and up tonine yeast cell components, wherein the cells of six yeast cellcomponents perform the basic functions of fixing atmospheric nitrogen,decomposing phosphorus-containing minerals or maintaining a balance ofphosphorus compounds, decomposing potassium-containing minerals,decomposing complex carbon-containing materials or compounds,overproducing growth factors, and overproducing ATP, and wherein thecells of the other component(s) perform the supplementary functions ofsuppressing growth of pathogenic microorganisms, decomposing undesirablechemicals, and reducing the odor of the organic substrate in thefertilizer composition.

In preferred embodiments, the present invention uses yeasts that arecommercially available and/or accessible to the public, such as but notlimited to Saccharomyces cerevisiae. Generally, the yeast cellcomponents of the invention are produced by culturing the pluralities ofyeast cells under activation conditions such that the abilities of thepluralities of cells to perform the functions are activated or enhanced.Accordingly, in another embodiment, the invention encompasses methods ofactivating or enhancing the abilities of yeast cells to perform one ofthe ten functions. The invention also relates to methods formanufacturing the fertilizer comprising mixing sludge with the yeastcells of the present invention, followed by drying and packing the finalproduct.

The invention further relates to methods for using the fertilizercompositions of the present invention. The biological fertilizercompositions of the present invention are used to support and enhancethe growth and maturation of a wide variety of plants.

4. BRIEF DESCRIPTION OF FIGURES

FIG. 1 Activation of yeast cells. 1 yeast cell culture; 2 container; 3electromagnetic field source.

FIG. 2. Formation of symbiosis-like relationships among strains ofyeasts. 4 electromagnetic field source for nitrogen-fixing yeasts; 5electromagnetic field source for P-decomposing yeasts; 6 electromagneticfield source for K-decomposing yeasts; 7 electromagnetic field sourcefor C-decomposing yeasts; 8 yeast cell culture; 9 container.

FIG. 3. Adaptation of yeast cells to a soil type. 10 electrode; 11container; 12 electrode; 13 yeast cell culture; 14 electromagnetic fieldsource; 15 temperature controller.

FIG. 4. Organic substrate grinding process. 16 organic raw material; 17crusher; 18 grinder; 19 organic substrate in powder form.

FIG. 5. Inorganic substrate grinding process. 20 inorganic raw material;21 crusher; 22 grinder; 23 inorganic substrate in powder form.

FIG. 6. Yeast fermentation process. 24 activated yeast cells; 25 tankfor culturing yeast cells, starch: water (35° C.)=1:2.5, semi-aerobicfermentation at 28 to 30° C.; 26 harvested culture.

FIG. 7. Mixing organic and inorganic raw materials. 27 inorganicmaterials; 28 starch; 29 organic materials; 30 mixer; 31 mixture; 32mixture to be transported to fertilizer production stage.

FIG. 8. Mixing yeast cells. 33 nitrogen-fixing yeasts 34 P-decomposingyeasts; 37 K-decomposing yeasts; 55 C-decomposing microbes; 35ATP-producing yeasts; 36 GF-producing yeasts; 52 pathogen-suppressingyeasts; 53 yeasts that decompose undesirable chemicals; 54 deodorizingyeasts; 38 mixture of yeasts; 56 mixture to be transported to fertilizerproduction stage.

FIG. 9. Fertilizer production process. 39 mixture of yeasts; 40 mixtureof organic and inorganic materials; 41 granulizer; 42 fertilizergranules.

FIG. 10. Drying process. 43 fertilizer granules; 44 first dryer; 45second dryer; 46 dried fertilizer.

FIG. 11. Cooling and packaging process. 47 dried fertilizer; 48 cooler;49 separator; 50 bulk bag filler; 51 final product.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides biological fertilizer compositions thatcomprise yeast cells and sludge. The present invention also providesmethods for manufacturing the biological fertilizer compositions as wellas methods for using the biological fertilizer compositions.

The biological fertilizer compositions of the invention can replacechemical/mineral fertilizers in supplying nitrogen (N), phosphorus (P),and potassium (K) to plants, especially crop plants. The inclusion ofsludge in the biological fertilizer compositions of the inventionprovide an environmentally acceptable and economic method for recyclingsludge.

According to the invention, the biological fertilizer compositionscomprise poultry manure and a plurality of yeast cell components. Eachyeast cell component is a population of yeast cells which comprises aplurality of yeast cells that are capable of performing a desiredfunction. The yeast cell components of the invention can provide thefollowing six basic functions: (1) fixation of atmospheric nitrogen; (2)decomposition of phosphorus minerals or compounds, or maintaining abalance of phosphorus compounds; (3) decomposition of potassium mineralsor compounds; (4) decomposition of complex or high molecular weightcarbon materials or compounds; (5) overproduction of growth factors; and(6) overproduction of ATP. The yeast cell components of the inventioncan provide the following supplementary functions: (7) suppression ofgrowth of pathogens, (8) degradation of undesirable chemicals, or (9)reducing the odor of organic materials.

In one embodiment, a biological fertilizer composition of the inventioncomprises (I) poultry manure; (II) at least one of the following yeastcell component: (a) a first yeast cell component comprising a firstplurality of yeast cells that fix nitrogen; (b) a second yeast cellcomponent comprising a second plurality of yeast cells that decomposephosphorus compounds; or (c) a third yeast cell component comprising athird plurality of yeast cells that decompose potassium compounds; and(III) at least one of the following: (d) a fourth yeast cell componentcomprising a fourth plurality of yeast cells that suppress the growth ofpathogenic microorganisms; (e) a fifth yeast cell component comprising afifth plurality of yeast cells that degrade antibiotics; or (f) a sixthyeast cell component comprising a sixth plurality of yeast cells thatreduce the odor of the biological fertilizer composition. Thus, abiological fertilizer composition of the invention comprises at leasttwo yeast cell components, one providing one of the three listed basicfunctions and one providing a supplementary function. In anotherembodiment, the biological fertilizer composition as described abovefurther comprises at least one of the following: (g) a seventh yeastcell component comprising a seventh plurality of yeast cells thatconvert complex carbon compounds to simple carbohydrates; (h) an eighthyeast cell component comprising an eighth plurality of yeast cells thatoverproduce growth factors; or (i) a ninth yeast cell componentcomprising a ninth plurality of yeast cells that overproduce adenosinetriphosphate. In preferred embodiments, the biological fertilizercompositions of the invention comprises yeast cell components thatprovide all six basic functions, plus at least one of the supplementaryfunctions. Thus, the preferred biological fertilizer compositionscomprise seven, eight or nine different yeast cell components.

The pluralities of the yeast cells of the invention can be added tosludge or existing organic fertilizers to improve their performance.

The sludge in the fertilizer compositions provides a source of nitrogen,hosphorus and potassium. Optionally, the fertilizer compositions mayinclude an inorganic component comprising minerals which provides anadditional source of phosphorous and/or potassium, and other mineralssuch as but not limited to calcium, magnesium, and sulfur; andmicronutrients, such as but not limited to boron, copper, iron,manganese, molybdenum, and zinc.

The biological fertilizer compositions of the present invention havemany advantages over mineral fertilizers and organic fertilizers.Because the biological fertilizer of the present invention utilizemetabolic activities of living yeasts to convert raw materials, such asatmospheric nitrogen, and phosphorus and potassium compounds in thesubstrate component, into plant nutrients, the conversion and release ofsuch nutrients by the yeast cells is regulated in part by the nutrientcontent of the soil. The nutrient content of the soil in turn depends inpart on both the environment and the changing needs of plants.Therefore, the release of plant nutrients by the biological fertilizercompositions is adaptable to the soil condition and can be sustainedover a period of time.

In addition to supplying nutrients to plants, the biological fertilizercompositions of the invention provide up to three supplementaryfunctions that mitigate some of the undesirable properties of sludgethat tend to restrict their use as organic fertilizers. The presence ofpathogenic bacteria in sludge poses a health risk to humans andlivestock. The biological fertilizer compositions can include acomponent of yeast cells that can suppress the proliferation ofpathogenic bacteria, thereby reducing the risk of infection, andcircumventing the need to use chemicals in controlling the spread ofsuch pathogens. Another yeast cell component that can be included in thecomposition is capable of reducing the odor of sludge, thus making itsinclusion in a fertilizer more acceptable. Yet another yeast cellcomponent can be included to degrade undesirable chemicals, such asantibiotic feed additives, which are found in sludge. Thesesupplementary functions generally lessen the adverse impact on theenvironment of using sludge in a fertilizer. The yeast cell componentsthat provide the supplementary functions can each be separately includedwith the other six yeast cell components that provide the basicfunctions, or in combination with each other and the other sixcomponents to provide the desired assortment of supplementary functions.

While the following terms are believed to have well-defined meanings inthe art, the following are set forth to facilitate explanation of theinvention.

As used herein, the term “nitrogen fixation” or “fixation of atmosphericnitrogen” encompasses biological processes in which molecular nitrogenor nitrogen in the atmosphere is converted into one or more nitrogenous(N) compounds, including but not limited to, ammonia, ammonium salts,urea, nitrites, and nitrates.

As used herein, the phrase “decomposition of phosphorus minerals orcompounds” refers to biological processes which convert phosphorus (P)compounds, such as but not limited to those water-insoluble phosphoruscompounds present in minerals, such as phosphate rock, into one or moredifferent phosphorus compound(s) which are biologically available ormore readily assimilable, i.e., usable for survival and/or growth, byplants and other yeasts. For example, the resulting phosphorus compoundsmay be more soluble in water or weak acid, and can thus be taken up bythe roots of plants. Non-limiting examples of biologically available orassimilable phosphorus compounds include various classes of phosphatessuch as H₂PO₄ ⁻ and HPO₄ ²⁻.

As used herein, the phrase “maintenance of a balance of phosphoruscompounds” refers to biological processes which convert biologicallyunavailable or water-insoluble phosphorus compounds into one or moredifferent phosphorus compound(s) which are more biologically availableor soluble in water, wherein the processes are sensitive to excess orthe lack of phosphorus (P) compounds in the local environment. Theconversion process is downregulated when the level of P compound is high(i.e., greater than about 180 ppm) and upregulated when level of Pcompound is low (i.e., greater than about 60 ppm)

As used herein, the phrase “decomposition of potassium minerals orcompounds” refers to biological processes which convert potassium (K)compounds, such as but not limited to those water-insoluble potassiumcompounds present in potassium-containing minerals and materials, intoone or more different potassium compound(s) which can be biologicallyavailable or more readily assimilable by plants and other yeasts. Forexample, the resulting potassium compounds may be more soluble in water,and can thus be taken up by the roots of plants.

As used herein, the phrase “decomposition of complex or high molecularweight carbon minerals, materials or compounds” refers to the biologicalconversion of a complex organic or inorganic carbon molecule (e.g.complex carbohydrates like cellulose and lignin into one or more carboncompound(s) which are of a lower molecular weight (e.g., simplecarbohydrates) and can be readily used for survival and/or growth byplants and yeasts. This process includes those reactions where longchains of carbon atoms in a polymeric carbon compound are cleaved.

As used herein, the term “growth factors” refers to molecules commonlyrequired for the growth of yeasts, including but not limited tovitamins, in particular, vitamin B complexes, e.g., vitamin B-1,riboflavin (vitamin B-2), vitamin B-12, niacin (B-3), pyridoxine (B-6),pantothenic acid (B-5); folic acid; biotin; para-aminobenzoic acid;choline; and inositol.

For the purpose of this invention, the above-described five functionstogether with the overproduction of growth factors and ATP are referredto as the basic functions.

As used herein, the phrase “suppressing the growth of pathogens” refersto a decrease or lack of increase in the number of pathogenicmicroorganisms present in a sample of sludge over a period of time, as aresult of the presence of the yeast cells of the invention in thesample. It is to be understood that in the absence of the yeast cells,the number of pathogens in the sample would increase naturally. Manysuch microorganisms cause diseases in humans and animals, and mayinclude bacteria such as Escherichia species, Salmonella species,Shigella species, Mycobacterium species, Staphylococcus species,Bacillus species, Streptococcus and Diplococcus species.

As used herein, the phrase “degradation of undesirable chemicals” refersto biological or biochemical processes which result in the conversion ofchemical compounds that are undesirable in a fertilizer to an inactiveform, such as the breakdowvn of such compounds into lower molecularweight compounds. Antibiotics are commonly present in organic materialsand such compounds are not desired in a fertilizer because of thepotential risk of ingestion by humans, for example, by eating vegetablesgrown using a fertilizer comprising contaminated organic material, andthe possible spread of antibiotic resistance in the environment. Manyantibiotics are added to animal feed to protect various farm animals,such as chicken, turkey, and swine, from bacterial and parasiticdiseases, and to promote growth. A significant amount of antibiotic feedadditive is excreted by the animals, and thus accumulates in manure andsludge. Many kinds of antibiotics have been used in animal operations,such as but not limited to aminoglycosides, tetracyclines, beta-lactams,glycopeptides, and macrolides. Examples of antibiotics approved for usein farms in United States include but are not limited to, bacitracinmethylene disalicylate, bacitracin zinc, bambermycins, oxytetracycline,chlortetracycline, penicillin, tylosin/sulfamethazine, roxarsone,nitrasone, monensin, lasalocid, carbodox, tiamulin, hygromycin B,nystatin, novobiocin, sulfadimethoxine, ormetroprim, lincomycin,fenbendazole, and virginiamycin. The presence and quantity of suchantibiotics in a composition can be determined by any methods known inthe art, for example, high performance liquid chromatography (HPLC).

As used herein, the phrase “reducing the odor of organic materials”refers to a process which results in a lower concentration of one ormore odorous compounds in sludge. Odorous compounds, such as but notlimited to hydrogen sulfide, ammonia, indole, skatole (i.e,3-methyl-1H-indole), p-cresol, and organic acids, are known tocontribute to the malodorous quality of manure. The concentration ofsuch malodorous compounds in sludge or in a sample of air in contactwith the manure can be determined by any method well known in the art,including but not limited to gas chromatography. Odor is a perception ofsmell by an organism with olfactory organs. A reduction of the intensityof the odor associated with sludge can be determined subjectively.Various methods and techniques are known to measure the intensity of anodor. One subjective measurement of odor intensity is to measure thedilution necessary so that the odor is imperceptible or doubtful to ahuman or animal test panel. Alternatively, a recognition threshold mayalso be used which is a higher concentration at which the character ofthe odor is recognized. Any methods and techniques for objectively orsubjectively determine the intensity of an odor can be used to monitorthe performance of the compositions and methods of the invention.

For the purpose of this invention, the suppression of growth ofpathogens, degradation of undesirable chemicals, and reduction of odorof organic materials are referred to as the supplementary functions oractivities.

The inventor discovered that, under various culture conditions, yeastscan be induced to exhibit seven different basic functions and threesupplementary functions. The culture condition determines the activitywhich is activated or enhanced in the cultured yeasts. The specificculture conditions for each of the ten functions are described indetails in sections 5.1 to 5.10 respectively.

According to the invention, a yeast cell component of the biologicalfertilizer composition is produced by culturing a plurality of yeastcells in an appropriate culture medium in the presence of an alternatingelectromagnetic field or multiple alternating electromagnetic fields inseries over a period of time. The culturing process allows yeast sporesto germinate, yeast cells to grow and divide, and can be performed as abatch process or a continuous process. As used herein, the terms“alternating electromagnetic field”, “electromagentic field” or “EMfield” are synonymous. An electromagnetic field useful in the inventioncan be generated by various means well known in the art. A schematicillustration of exemplary setups are depicted respectively in FIG. 1. Anelectromagnetic field of a desired frequency and a desired fieldstrength is generated by an electromagnetic wave source (3) whichcomprises one or more signal generators that are capable of generatingelectromagnetic waves, preferably sinusoidal waves, and preferably inthe frequency range of 30 MHz-3000 MHz. Such signal generators are wellknown in the art. Signal generators capable of generating signal with anarrower frequency range can also be used. If desirable, a signalamplifier can also be used to increase the output signal, and thus thefield strength.

The electromagnetic field can be applied to the culture by a variety ofmeans including placing the yeast cells in close proximity to a signalemitter connected to a source of electromagnetic waves. In oneembodiment, the electromagnetic field is applied by signal emitters inthe form of electrodes that are submerged in a culture of yeast cells(1). In a preferred embodiment, one of the electrodes is a metal plate,and the other electrode comprises a plurality of wires configured insidethe container (2) so that the energy of the electromagnetic field can beevenly distributed in the culture. The number of electrode wires useddepends on both the volume of the culture and the diameter of the wire.For example, for a culture having a volume of 5000 ml, one electrodewire having a diameter of between 0.1 to 1.2 mm can be used for each 100ml of culture; for a culture having a volume greater than 1000 l, oneelectrode wire having a diameter of between 3 to 30 mm can be used foreach 1000 l of culture.

In preferred embodiments, yeasts of the genera of Saccharomyces,Schizosaccharomyces, Sporobolomyces, Torulopsis, Trichosporon,Wickerhamia, Ashbya, Blastomyces, Candida, Citeromyces, Crebrothecium,Cryptococcus, Debaryomyces, Enclomycopsis; Geotrichum, Hansenula,Kloeckera, Lipomyces, Pichia, Rhodosporidium, and Rhodotorula can beused in the invention.

Non-limiting examples of yeast strains include Saccharomyces cerevisiaeHansen, ACCC2034, ACCC2035, ACCC2036, ACCC2037, ACCC2038, ACCC2039,ACCC2040, ACCC2041, ACCC2042, AS2.1, AS2.4, AS2.11, AS2.14, AS2.16,AS2.56, AS2.69, AS2.70, AS2.93, AS2.98, AS2.101, AS2.109, AS2.110,AS2.112, AS2.139, AS2.173, AS2.174, AS2.182, AS2.196, AS2.242, AS2.336,AS2.346, AS2.369, AS2.374, AS2.375, AS2.379, AS2.380, AS2.382, AS2.390,AS2.393, AS2.395, AS2.396, AS2.397, AS2.398, AS2.399, AS2,400, AS2.406,AS2.408, AS2.409, AS2.413, AS2.414, AS2.415, AS2.416, AS2.422, AS2.423,AS2.430, AS2.431, AS2.432, AS2.451, AS2.452, AS2.453, AS2.458, AS2.460,AS2.463, AS2.467, AS2.486, AS2.501, AS2.502, AS2.503, AS2.504, AS2.516,AS2.535, AS2.536, AS2.558, AS2.560, AS2.561, AS2.562, AS2.576, AS2.593,AS2.594, AS2.614, AS2.620, AS2.628, AS2.631, AS2.666, AS2.982, AS2.1190,AS2.1364, AS2.1396, IFFI 1001, IFFI 1002, IFFI 1005, IFFI 1006, IFFI1008, IFFI 1009, IFFI 1010, IFFI 1012, IFFI 1021, IFFI 1027, IFFI 1037,IFFI 1042, IFFI 1043, IFFI 1045, IFFI 1048, IFFI 1049, IFFI 1050, IFFI1052, IFFI 1059, IFFI 1060, IFFI 1063, IFFI 1202, IFFI 1203, IFFI 1206,IFFI 1209, IFFI 1210, IFFI 1211, IFFI 1212, IFFI 1213, IFFI 1215, IFFI1220, IFFI 1221, IFFI 1224, IFFI 1247, IFFI 1248, IFFI 1251, IFFI 1270,IFFI 1277, IFFI 1287, IFFI 1289, IFFI 1290, IFFI 1291, IFFI 1291, IFFI1292, IFFI 1293, IFFI 1297, IFFI 1300, IFFI 1301, IFFI 1302, IFFI 1307,IFFI 1308, IFFI 1309, IFFI 1310, IFFI 1311, IFFI 1331, IFFI 1335, IFFI1336, IFFI 1337, IFFI 1338, IFFI 1339, IFFI 1340, IFFI 1345, IFFI 1348,IFFI 1396, IFFI 1397, IFFI 1399, IFFI 1411, IFFI 1413; Saccharomycescerevisiae Hansen Var. ellipsoideus (Hansen) Dekker, ACCC2043, AS2.2,AS2.3, AS2.8, AS2.53, AS2.163, AS2.168, AS2.483, AS2.541, AS2.559,AS2.606, AS2.607, AS2.611, AS2.612; Saccharomyces chevalieriGuillermond, AS2.131, AS2.213; Saccharomyces delbrueckii, AS2.285;Saccharomyces delbrueckii Lindner var. mongolicus Lodder et van Rij,AS2.209, AS2.1157; Saccharomyces exiguus Hansen, AS2.349, AS2.1158;Saccharomyces fermentati (Saito) Lodder et van Rij, AS2.286, AS2.343;Saccharomyces logos van laer et Denamur ex Jorgensen, AS2.156, AS2.327,AS2.335; Saccharomyces mellis Lodder et Kreger Van Rij, AS2.195;Saccharomyces microellipsoides Osterwalder, AS2.699; Saccharomycesoviformis Osterwalder, AS2.100; Saccharomyces rosei (Guillermond) Lodderet kreger van Rij, AS2.287; Saccharomyces rouxii Boutroux, AS2.178,AS2.180, AS2.370, AS2.371; Saccharomyces sake Yabe, ACCC2045; Candidaarborea, AS2.566; Candida Krusei (Castellani) Berkhout, AS2.1045;Candida lambica(Linciner et Genoud) van.Uden et Buckley, AS2.1182;Candida lipolytica (Harrison) Diddens et Lodder, AS2.1207, AS2.1216,AS2.1220, AS2.1379, AS2.1398, AS2.1399, AS2.1400; Candida parapsilosis(Ashford) Langeron et Talice, AS2.590; Candida parapsilosis (Ashford) etTalice Var. intermedia Van Rij et Verona, AS2.491; Candida pulcherriman(Lindner) Windisch, AS2.492; Candida rugousa (Anderson) Diddens etLoddeer, AS2.511, AS2.1367, AS2.1369, AS2.1372, AS2.1373, AS2.1377,AS2.1378, AS2.1384; Candida tropicalis (Castellani) Berkout, ACCC2004,ACCC2005, ACCC2006, AS2.164, AS2.402, AS2.564, AS2.565, AS2.567,AS2.568, AS2.617, AS2.1387; Candida utilis Henneberg Lodder et KregerVan Rij, AS2.120, AS2.281, AS2.1180; Crebrothecium ashbyii (Guillermond)Routein, AS2.481, AS2.482, AS2.1197; Geotrichum candidum Link, ACCC2016,AS2.361, AS2.498, AS2.616, AS2.1035, AS2.1062, AS2.1080, AS2.1132,AS2.1175, AS2.1183; Hansenula anomala (Hansen) H et P sydow, ACCC2018,AS2.294, AS2.295, AS2.296, AS2.297, AS2.298, AS2.299, AS2.300, AS2.302,AS2.338, AS2.339, AS2.340, AS2.341, AS2.470, AS2.592, AS2.641, AS2.642,AS2.635, AS2.782, AS2.794; Hansenula arabitolgens Fang, AS2.887;Hansenula jadinii Wickerham, ACCC2019; Hansenula saturnus (Klocker) H etP sydow, ACCC2020; Hansenula schneggi (Weber) Dekker, AS2.304; Hansenulasubpelliculosa Bedford, AS2.738, AS2.740, AS2.760, AS2.761, AS2.770,AS2.783, AS2.790, AS2.798, AS2.866; Kloeckera apiculata (Reess emend.Klocker) Janke, ACCC2021, ACCC2022, ACCC2023, AS2.197, AS2.496, AS2.711,AS2.714; Lipomyces starkeyi Lodder et van Rij, ACCC2024, AS2.1390;Pichia farinosa (Lindner) Hansen, ACCC2025, ACCC2026, AS2.86, AS2.87,AS2.705, AS2.803; Pichia membranaefaciens Hansen, ACCC2027, AS2.89,AS2.661, AS2.1039; Rhodosporidium toruloides Banno, ACCC2028;Rhodotorula glutinis (Fresenius) Harrison, ACCC2029, AS2.280, ACCC2030,AS2.102, AS2.107, AS2.278, AS2.499, AS2.694, AS2.703, AS2.704, AS2.1146;Rhodotorula minuta (Saito) Harrison, AS2.277; Rhodotorula rubar (Demme)Lodder, ACCC2031, AS2.21, AS2.22, AS2.103, AS2.105, AS2.108, AS2.140,AS2.166, AS2.167, AS2.272, AS2.279, AS2.282; Saccharomycescarlsbergensis Hansen, ACCC2032, ACCC2033, AS2.113, AS2.116, AS2.118,AS2.121, AS2.132, AS2.162, AS2.189, AS2.200, AS2.216, AS2.265, AS2.377,AS2.417, AS2.420, AS2.440, AS2.441, AS2.443, AS2.444, AS2.459, AS2.595,AS2.605, AS2.638, AS2.742, AS2.745, AS2.748, AS2.1042; Saccharomycesuvarum Beijer, IFFI 1023, IFFI 1032, IFFI 1036, IFFI 1044, IFFI 1072,IFFI 1205, IFFI 1207; Saccharomyces willianus Saccardo, AS2.5, AS2.7,AS2.119, AS2.152, AS2.293. AS2.381, AS2.392, AS2.434, AS2.614. AS2.1189:Saccharomyces sp., AS2.311 ; Saccharomyces ludwigii Hansen, ACCC2044,AS2.243, AS2.508; Saccharomyces sinenses Yue AS2.1395;Schizosaccharomyces octosporus Beijerinck, ACCC 2046, AS2.1148;Schizosaccharomyces pombe Linder, ACCC2047, ACCC2048, AS2.248, AS2.249,AS2.255, AS2.257, AS2.259, AS2.260, AS2.274, AS2.994, AS2.1043,AS2.1149, AS2.1178, IFFI 1056; Sporobolomyces roseus Kluyver et vanNiel, ACCC 2049, ACCC 2050, AS2.619, AS2.962, AS2.1036, ACCC2051,AS2.261, AS2.262; Torulopsis candida (Saito) Lodder, ACCC2052, AS2.270;Torulopsis famta (Harrison) Lodder et van Rij, ACCC2053, AS2.685;Torulopsis globosa (Olson et Hammer) Lodder et van Rij, ACCC2054,AS2.202; Torulopsis inconspicua Lodder et van Rij, AS2.75; Trichosporonbehrendii Lodder et Kreger van Rij, ACCC2055, AS2.1193; Trichosporoncapitatum Diddens et Lodder, ACCC2056, AS2.1385; Trichosporoncutaneum(de Beurm et al.)Ota, ACCC2057, AS2.25, AS2.570, AS2.571,AS2.1374; Wickerhamia fluoresens (Soneda) Soneda, ACCC2058, AS2.1388.

Certain yeast species that can be activated or induced according to thepresent invention and are included in the present invention are known tobe pathogenic to human and/or other living organisms, for example,Ashbya gossypii; Blastomyces dermatitidis; Candida albicans; Candidaparakrusei; Candida tropicalis; Citeromyces matritensis; Crebrotheciumashbyii; Cryptococcus laurentii; Cryptococcua neoformans; Debaryomyceshansenii; Debaryomyces kloeckeri; Debaryomyces sp.; Endomycopsisfibuligera. Under certain circumstances, it may be less preferable touse such pathogenic yeasts in the biological fertilizer of theinvention, for example, if such use in an open field may endanger thehealth of human and/or other living organisms.

Yeasts of the Saccharomyces genus are generally preferred. Among strainsof Saccharomyces cerevisiae, Saccharomyces cerevisiae Hansen is apreferred strain. The most preferred strains of yeast are Saccharomycescerevisiae strains having accession numbers AS2.504, AS2.558, AS2.413,AS2.397, AS2.69, AS2.109, AS2.607, AS2.516, AS2.561, AS2.422, AS2.393,AS2.631, AS2.982, AS2.560, AS2.467, AS2.415, AS2.375, AS2.628, AS2.1190,AS2.562, AS2.463, AS2.409, AS2.379, AS2.666, AS2.631, AS2.182, AS2.431,AS2.606, AS2.53, AS2.611, AS2.414, AS2.576, AS2.483, IFFI 1211, IFFI1293, IFFI 1308, IFFI 1210, IFFI 1213, IFFI 1307, IFFI 1206, IFFI 1052,IFFI 1301, IFFI 1291, IFFI 1202, IFFI 1021, IFFI 1059, IFFI 1052, IFFI1441, IFFI 1008, IFFI 1220, IFFI 1302, and IFFI 1023 as deposited at theChina General Microbiological Culture Collection Center (CGMCC).

Generally, yeast strains useful for the invention can be obtained fromprivate or public laboratory cultures, or publically accessible culturedeposits, such as the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209 and the China GeneralMicrobiological Culture Collection Center (CGMCC), China Committee forCulture Collection of Microorganisms, Institute of Microbiology, ChineseAcademy of Sciences, Haidian, P.O. Box 2714, Beijing, 100080, China.

The following yeast strains are preferred for making the P-balancingyeasts of the invention: AS2.558, AS2.118, AS2.103, AS2.132, AS2.121,AS2.189, AS2.216, AS2.265, AS2.417, AS2.420, AS2.200, AS2.162, AS2.440,AS2.277, AS2.441, AS2.443, AS2.444, AS2.605, AS2.595, AS2.638, AS2.742,AS2.748, AS2.14, AS2.16, AS2.56, AS2.69, AS2.70, AS2.109, AS2.112,AS2.375, AS2560, AS2.561, AS2.562, AS2.559, AS2.501, AS2.502, AS2.503,AS2.504, IFFI1001, IFFI1002, IFFI1005, IFFI1006, IFFI1008, IFFI1009,IFFI1010, IFFI1012, IFFI1021, IFFI1027, IFFI1037, IFFI1042, IFFI1060,IFFI1063, IFFI1202, IFFI1203, IFFI1206, IFFI1209, IFFI1210, IFFI1211,IFFI1212, IFFI1213, IFFI1215, IFFI1220, IFFI1221, IFFI1224, IFFI1247,IFFI1248, IFFI1251, IFFI1270, IFFI1277, IFFI1287, IFFI1289, IFFI1290,IFFI1291, IFFI1292, IFFI1293, IFFI1297, IFFI1300, IIFFI1301, IFFI1207,IFFI1307, IFFI1308, IFFI1309, IFFI1310, IFFI1311, IFFI1331, IFFI1335,IIFFI1336, IFFI1337, IFFI1338, IFFI1340, IFFI1339, IFFI1345, IFFI1396,IFFI1399, IFFI1411, IFFI1413, IFFI1023, IFFI1032, IFFI1036, IFFI1044,and IFFI1207.

Although it is preferred, the preparation of the yeast cell componentsof the invention is not limited to starting with a pure strain of yeast.Each yeast cell component may be produced by culturing a mixture ofyeast cells of different species or strains. The constituents of a yeastcell component can be determined by standard yeast identificationtechniques well known in the art.

Some yeasts may perform one of the desired functions more efficientlythan others. The table below lists the species and accession numbers ofvarious yeast strains and the preferred functions for which therespective strains are stimulated by the methods of the invention.

The ability and efficiency of any species or strain of yeast to performany one of the ten desired functions before or after culturing under theconditions of the invention can readily be tested by methods known inthe art. For example, the amount of nitrogen fixed can be determined bya modified acetylene reduction method as described in U.S. Pat. No.5,578,486 which is incorporated herein by reference in its entirety. Themodified acetylene reduction method determines the amount of nitrogenfixed by measuring the decrease in molecular nitrogen in a volume ofair. The amount of nitrogen fixed can also be determined by measurementof the ammonia and nitrates produced by the yeast cells (see, forexample. Grewling et al., 1965, Cornell Agr Exp Sta Bull 960:22-25). Theamount of phosphorus available to plants as a result of conversion frominsoluble or biologically-unavailable phosphorus compounds can bedetermined by the molybdenum blue method (see, for example, Murphy etal., 1962, Analytica Chimica Acta 27:31-36) or the UV absorption method;whereas the amount of available potassium converted from insoluble orbiologically-unavailable potassium compounds can be determined, forexample, by flame atomic absorption spectroscopy (see, for example,Puchyr, et al., 1986, J. Assoc. Off. Anal. Chem. 69:868-870). Theability of the yeasts to supply biologically available N, P, and K afterthe biological fertilizer composition has been added to soil can betested by many techniques known in the art. For example, plant-availableammonia, nitrates, P, and K produced by the yeast cells in soil can beextracted and quantitatively analyzed by the Morgan soil test system(see, for example, Lunt et al., 1950, Conn Agr Exp Sta Bull 541).

Methods well known in the art can be used for detecting and analyzingvarious organic molecules in sludge, including HPLC. Similarly, methodswell known in the art can be used for detecting and counting the numberof viable microorganisms and the total number of microorganisms in asample.

Without being bound by any theory or mechanism, the inventor believesthat the culture conditions activate and/or enhance the expression of agene or a set of genes in a yeast cell such that the cell becomes activeor more efficient in performing certain metabolic activities which leadto the respective desired results.

The term “sludge” as used herein broadly encompasses any solid matterthat has settled out of suspension in the course of sewage storageand/or treatment, for example, residues in a waste lagoon, in an urbansewage treatment plant. The term also include semi-solid matters, andmixtures of effluent and sediments. The term thus encompasses sludgehaving a wide range of viscosity, density, and water content, as well assludge which has been partially processed or stabilized.

Optionally, an inorganic substrate component can be included in thebiological fertilizer compositions of the invention. The inorganicsubstrate component can include but not limited to phosphate rock orrock phosphate, apatite, phosphorite, sylvinite, halite, carnalitite,and potassium mica.

Due to the variation of constituents in sludge, it may be desirable tosubject a sample of a batch of sludge to analysis to determine theamount of plant nutrient present. Methods of soil analysis well known inthe art can be used to measure the amount of N, P, K, calcium,magnesium, zinc, iron, manganese, copper, sodium and sulfur in thesludge.

In various embodiments, the biological fertilizer compositions of thepresent invention each comprises at least seven yeast cell componentscapable of performing six basic functions plus at least one of thesupplementary functions. In a most preferred embodiment, the biologicalfertilizer compositions comprise nine yeast cell components, in whichcase the six basic functions and all three supplementary functions areprovided. It will be understood that alternative formulations are alsocontemplated.

In one particular embodiment of the invention, when a batch of sludgethat is relatively rich in biologically-available phosphorus is used,the biological fertilizer composition can be formulated to compriseyeast cells that can maintain a balance of phosphorus compounds insteadof yeast cells that decompose phosphorus-containing minerals orcompounds. Moreover, if desired, the biological fertilizer compositionmay comprise lesser quantities of one or more of the above-describedyeast cell components that supply one of the six basic functions. Forexample, if the biological fertilizer composition is to be used in soilthat is rich in potassium, the biological fertilizer composition can beformulated to comprise lesser amount of the yeast cells that candecompose potassium-containing minerals or compounds.

In another embodiment of the invention, where the yeast cells of thevarious yeast cell components are present in a mixture, the yeast cellscan be cultured under certain conditions such that the yeast cells withdifferent functions can supply each other with and/or rely on each otherfor nutrients and growth factors. As a result, a symbiosis-likerelationship is established among the various yeast cell components inthe fertilizer compositions of the invention. This culturing process isoptional but can improve the stability and efficiency of thecompositions such that the resulting fertilizer is made more suitablefor long term use in natural soil environments. The culturing conditionsfor this optional process are described in Section 5.11.

In yet another embodiment of the invention, the yeast cells may also becultured under certain conditions so as to adapt the yeast cells to aparticular type of soil. This culturing process is optional, and can beapplied to each yeast cell component separately or to a mixture of yeastcell components. The result is better growth and survival of the yeastcells in a particular soil environment. The culturing conditions forthis optional process are described in Section 5.12.

As used herein, the biological fertilizer composition supports orenhances plant growth, if in the presence of the biological fertilizerin the soil, or applied to the roots, stems, leaves or other parts ofthe plant, the plant or a part of the plant gains viability, size,weight, rate of germination, rate of growth, or rate of maturation.Thus, the biological fertilizer compositions have utility in any kind ofagricultural, horticultural, and forestry practices. The biologicalfertilizer compositions can be used for large scale commercial farming,in open fields or in greenhouse, or even ill interiors for decorativeplants. Preferably, the biological fertilizer is used to enhance thegrowth of crop plants, such as but not limited to cereal crops,vegetable crops, fruit crops, flower crops, and grass crops. Forexample, the biological fertilizer compositions may be used with wheat,barley, corn, soybean, rice, oat, potato, apple, orange, tomato, melon,cherry, lemon, lettuce, carrot, sugar cane, tobacco, cotton, etc.

The biological fertilizer compositions of the invention may be appliedin the same manner as conventional fertilizers. As known to thoseskilled in the relevant art, many methods and appliances may be used. Inone embodiment, culture broths of the yeast strains of the presentinvention are applied directly to soil or plants. In another embodiment,dried powders of the yeast strains of the present invention are appliedto soil or plants. In yet another embodiment, mixtures of the yeast cellcomponents and organic substrate components of the present invention areapplied to soil or plants. The biological fertilizer compositions may beapplied to soil, by spreaders, sprayers, and other mechanized meanswhich may be automated. The biological fertilizer compositions may beapplied directly to plants, for example, by soaking seeds and/or roots,or spraying onto leaves. Such application may be made periodically, suchas once per year, or per growing season, or more frequently as desired.The biological fertilizer compositions of the invention can also be usedin conjunction or in rotation with other types of fertilizers.

In one preferred embodiment, the biological fertilizer composition ofthe invention, i.e., yeasts of the invention mixed with sludge ingranular form, is used as a basal fertilizer which is applied into thesoil at the depth of the major root system of the crop. Prior toapplication, the ground should be loosened and clear of weeds. Thebiological fertilizer composition can be spread evenly onto the ground,added to holes or long furrows in the ground. For existing fruit trees,a circular furrow of about 5 to 30 cm deep is dug into which thebiological fertilizer composition of the invention is added. The ground,holes, or furrows containing the biological fertilizer composition canthen be covered with soil and watered throughly. After 3 to 7 days, thearea is ready for planting or sowing. For rice, the ground is floodedwith water for 3 to 7 days before planting the seedlings. If used insandy soil with a shallow root system, a depth of 5 to 15 cm is used;with a deep root system, 5 to 25 cm is recommended. In clay soil with ashallow root system, a depth of 2 to 10 cm is used; with a deep rootsystem, 2 to 15 cm is recommended. The desired effect is that thebiological fertilizer composition is contact with or in very closeproximity to the roots of the plants. Preferably, after application ofthe fertilizer and/or planting, the soil is not disturbed. Generally,the operation temperature of the fertilizer is between 5° C. to 45° C.,optimally between 16° C. to 30° C.; the preferred pH range is between5.5 to 8.5, and optimally between 6.5 to 7.5.

Recommended Dosage Crop Amount of Biological Fertilizer Vegetables(short-growing) 600-900 kg/ha Vegetable (long-growing) 900-1200 kg/haGround vegetable 900-1350 kg/ha Solanaceous fruit 900-1350 kg/ha Root &Tuber vegetable 750-900 kg/ha Bulb vegetable 900-1200 kg/ha Legume600-1050 kg/ha Fruit Trees 2-5 kg/tree Paddy Rice 600-900 kg/ha Wheat &Corn 750-1200 kg/ha Cotton & Peanut 600-1200 kg/ha

Described respectively in Sections 5.1-5.10 are the yeast cellcomponents used for nitrogen fixation, phosphorus compounddecomposition, potassium compound decomposition, complex carbon compounddecomposition, growth factors production, ATP production, pathogensuppression, degradation of undesirable chemicals, and reduction ofodor. Methods for preparing each yeast cell components are described.Section 5.11 describes the methods for establishing a symbiosis-likerelationship among yeast strains in a fertilizer composition of theinvention. Section 5.12 describes methods for adapting yeast cells ofthe invention to a particular type of soil. Section 5.13 describes themanufacture of the biological fertilizer compositions of the invention.Methods for the preparation of organic substrates and for themanufacture of the biological fertilizer, including mixing, drying,cooling, and packing, are also described. In various embodiments of theinvention, standard techniques for handling, transferring, and storingyeasts are used. Although it is not necessary, sterile conditions orclean environments are desirable when carrying out the processes of theinvention.

5.1. Nitrogen-Fixing Yeast Cell Component

Nitrogen fixation is a process whereby atmospheric nitrogen is convertedinto ammonia and nitrates. Close to 800 species of naturally occurringmicroorganisms, mostly bacteria and cyanobacteria, from more than 70genera have been found to be able to fix nitrogen. Some of thenitrogen-fixing microorganisms, such as Rhizoboum, form symbioticassociation with plants, especially in the root of legumes. Others, suchas Azotobacter, are free-living and capable of fixing nitrogen in soil.

In the present invention, the ability of a yeast to fix nitrogen isactivated or enhanced, and the resulting nitrogen-fixing yeast cells canbe used as a component of the biological fertilizer compositions of theinvention.

According to the invention, yeast cells that have an enhanced ability tofix nitrogen are prepared by culturing the cells in the presence of anelectromagnetic field in an appropriate culture medium. The frequency ofthe electromagnetic field for activating or enhancing nitrogen fixitionin yeasts can generally be found within the range of 800 MHz-1000 MHz.After the yeast cells have been cultured for a sufficient period oftime, the cells can be tested for their ability to fix nitrogen bymethods well known in the art.

The method of the invention for making the nitrogen-fixing yeast cellsis carried out in a liquid medium. The medium contains sources ofnutrients assimilable by the yeast cells. In general, carbohydrates suchas sugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 1%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

Among the inorganic salts which can be incorporated in the culture mediaare the customary salts capable of yielding sodium, potassium, calcium,phosphate, sulfate, carbonate, and like ions. Non-limiting examples ofnutrient inorganic salts are CaCO₃, KH₂PO₄, MgSO₄, NaCl, and CaSO₄.

TABLE 1 Composition for a culture medium for nitrogen-fixing yeastMedium Composition Quantity KH₂PO₄ 0.2 g K₂HPO₄ 0.2 g MgSO₄.7H₂O 0.25 gCaCO₃.5H₂O 3.5 g CaSO₄.2H₂O 0.5 g NaCl 0.25 g Yeast extract paste 0.3 gSucrose 12.0 g Distilled water or autoclaved water 1000 ml

It should be noted that the composition of the media provided in Table 1is not intended to be limiting. Various modifications of the culturemedium may be made by those skilled in the art, in view of practical andeconomic considerations, such as the scale of culture and local supplyof media components.

The process can be initiated by inoculating 100 ml of medium with 1 mlof an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

The EM field(s), which can be applied by any means known in the art, caneach have a frequency in the range of about 800 to about 1000 MHz,preferably in the range of 840.000 to 916.000 MHz. For example andwithout being limited by such examples, each EM field can have afrequency at about 840, 845, 850, 855, 860, 865, 870, 875, 880, 885,890, 895, 900, 905, 910, 915, or 920 MHz. The field strength of the EMfield(s) is in the range of 10 to 200 mV/cm. If a series of EM fieldsare applied, the EM fields can each have a different frequency withinthe stated range, or a different field strength within the stated range,or different frequency and field strength within the stated ranges. In apreferred embodiment, the EM field(s) at the beginning of a series havea lower EM field strength than later EM field(s), such that the yeastcell culture are exposed to EM fields of progressively increasing fieldstrength. Although any practical number of EM fields can be used withina series, it is preferred that the yeast culture be exposed to a totalof 2, 3, 4, 5, 6, 7, or 8 different EM fields in a series.

Although the yeast cells will become activated even after a few hours ofculturing in the presence of the EM field(s), and the yeast cells can becultured in the presence of the EM field(s) for an extended period oftime (e.g., one or more weeks), it is generally preferred that theactivated yeast cells be allowed to multiply and grow in the presence ofthe EM field or EM fields for a total of about 140-280 hours.

For example, using an exemplary apparatus as depicted in FIG. 1, aninitial EM field in the range of 10-20 mV/cm, usually at about 12.5mV/cm is used. After this first period of culture, the yeast cells arefurther incubated under substantially the same conditions for anotherperiod, except that the EM field strength is increased to a higher levelin the range of 50-200 mV/cm, usually to about 125 mV/cm. The process ofthe invention is carried out at temperatures ranging from about 23° to30° C.; however, it is preferable to conduct the process at 25° to 28°C. The culturing process may preferably be conducted under conditions inwhich the concentration of dissolved oxygen is between 0.025 to 0.8mol/m³, preferably 0.4 mol/m³. The oxygen level can be controlled by anyconventional means known to one skilled in the art, including but notlimited to stirring and/or bubbling.

At the end of the culturing process, the nitrogen-fixing yeast cells maybe recovered from the culture by various methods known in the art, andstored at a temperature below about 0° C. to 4° C. The nitrogen-fixingyeast cells may also be dried and stored in powder form.

Any methods known in the art can be used to test the activated yeastcells for their ability to fix nitrogen. For example, a modifiedacetylene reduction method for measuring nitrogen fixed bymicroorganisms is used to evaluate the nitrogen-fixing capability of theprepared yeast. The modified acetylene reduction method is described inU.S. Pat. No. 5,578,486 which is incorporated herein by reference in itsentirety. An alternative method based on 15-N can also be used.

The ability of the yeasts of the invention in fixing nitrogen can bedemonstrated by the following two methods:

One ml of activated yeast strain AS2.628 (2-5×10⁷) was cultured in 1000ml of Ashby medium at 28° C. in the presence of a series of 8 EM fieldsin the order stated: 855 MHz at 14 mV/cm for 5 hours; 865 MHz at 14mV/cm for 5 hours; 875 MHz at 14 mV/cm for 5 hours; 885 MHz at 14 mV/cmfor 5 hours; 855 MHz at 120 mV/cm for 30 hours; 865 MHz at 120 mV/cm for30 hours; 875 MHz at 120 mV/cm for 30 hours; 885 MHz at 120 mV/cm for 30hours. In a separate container, as control, 1 ml of non-activated yeastwas cultured under the same conditions without the EM fields. Afterculturing, the 1000 ml of yeast cells are mixed with 3000 g sterilizedcoal dust powder, and then dried at less than 70° C. until the moisturecontent is less than 5%. The end product in powder form (0.1 g) wassealed with 10 ml of Ashby medium in a 100 ml culture flask (5 flasksfor each were used in the experiment). 10 ml of air was removed from theflasks by a syringe and replaced with 10 ml of acetylene (>99% purity).The culture flasks were incubated at 28° C. for 24-120 hours and theamount of acetylene reduced was measured by gas chromatography. Therewas no significant reduction of acetylene in the control containingnon-activated yeasts.

Alternatively, the isotopic nitrogen dilution method can be used. Theend product in powder form (0.1 g of non-activated and 0.1 g ofactivated yeasts) were cultured separately for 96 hours at 28° C. Theamount of nitrogen fixed by each was determined and compared. The amountof nitrogen fixed by activated yeasts was greater than 3.5 mg/g of thedried powder. The control containing non-activated yeasts did not showany significant fixation of nitrogen.

5.2. Phosphorus-Decomposing Yeast Cell Component

The phosphorus compound-decomposing (P-decomposing) yeast of theinvention converts insoluble or biologically-unavailablephosphorus-containing substances, such as phosphate rock, into solublephosphorous compounds so that they become available to plants.

In the present invention, the ability of yeasts to decompose insolublephosphorus-containing substances is activated or enhanced, and theresulting P-decomposing yeast cells can be used as a component of thebiological fertilizer compositions of the invention.

In various embodiments, P-decomposing yeast cells are employed in thecompositions of the invention when the level of soluble orbiologically-available phosphorous is low in the organic substrate (forexample, cattle manure, swine manure, sludge and garbage).

According to the invention, yeast cells that are capable ofP-decomposing are prepared by culturing the cells in the presence of anelectromagnetic field in an appropriate culture medium. The frequency ofthe electromagnetic field for activating or enhancing P-decomposition inmicrobes can generally be found in the range of 300 MRz to 500 MHz.After the cells have been cultured for a sufficient period of time, thecells can be tested for their ability to decompose phosphorus-containingsubstances by methods well known in the

The method of the invention for making the P-decomposing yeast cells iscarried out in a liquid medium. The medium contains sources of nutrientsassimilable by the yeast cells. In general, carbohydrates such assugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 1.5%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

Among the inorganic salts which can be incorporated in the culture mediaare the customary salts capable of yielding sodium, potassium, calcium,sulfate, carbonate, and like ions. Non-limiting examples of nutrientinorganic salts are CaCO₃, MgSO₄, NaCl, and CaSO₄. Non-biologicallyavailable forms of phosphorus-containing substances in a suitable formare also included in the media as dried organic substrate. Non-limitingexamples of dried organic substrate include manure, sludge and garbageof ≧150 mesh. Other insoluble phosphorus-containing substances can alsobe used either separately or in combination.

TABLE 2 Composition for a culture medium for P-decomposing yeast MediumComposition Quantity Sucrose 15 g NaCl 1.2 g MgSO₄.7H₂O 0.2 g CaCO₃.5H₂O3.0 g CaSO₄.2H₂O 0.3 g KNO₃ 0.3 g Yeast extract paste 0.5 g Dried sludge1.2 g to 2.4 g: Powder of > 150 mesh Autoclaved water 1000 ml

It should be noted that the composition of the media provided in Table 2is not intended to be limiting. Various modifications of the culturemedium may be made by those skilled in the art, in view of practical andeconomic considerations such as the scale of culture and local supply ofmedia components.

The process can be initiated by inoculating 100 ml of medium with 1 mlof an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

The EM field(s), which can be applied by any means known in the art, caneach have a frequency in the range of about 300 to about 500 MHz,preferably in the range of 340.000 to 435.000 MHz. For example andwithout being limited by such examples, each EM field can have afrequency at about 340, 345, 350, 355, 360, 365, 370, 375, 375, 380,385, 390, 395, 400, 405, 410, 415, 420, 425, 430 or 435 MHz. The fieldstrength of the EM field(s) is in the range of 10 to 200 mV/cm. If aseries of EM fields are applied, the EM fields can each have a differentfrequency within the stated range, or a different field strength withinthe stated range, or different frequency and field strength within thestated ranges. In a preferred embodiment, the EM field(s) at thebeginning of a series have a lower EM field strength than later EMfield(s), such that the yeast cell culture are exposed to EM fields ofprogressively increasing field strength. Although any practical numberof EM fields can be used within a series, it is preferred that the yeastculture be exposed to a total of 2, 3, 4, 5, 6, 7, or 8 different EMfields in a series.

Although the yeast cells will become activated even after a few hours ofculturing in the presence of the EM field(s), and the yeast cells can becultured in the presence of the EM field(s) for an extended period oftime (e.g., one or more weeks), it is generally preferred that theactivated yeast cells be allowed to multiply and grow in the presence ofthe EM field or EM fields for a total of about 140-280 hours.

For example, using an exemplary apparatus as depicted in FIG. 1, aninitial field strength in the range of 10-20 mV/cm, usually at about12.5 mV/cm is used. After this first period of culture, the yeast cellsare further incubated under substantially the same conditions foranother period, except that the EM field strength is increased to ahigher level in the range of 50-200 mV/cm, Usually to about 125 mV. Theprocess of the invention is carried out at temperatures ranging fromabout 23° to 30° C.; however, it is preferable to conduct the process at25° to 28° C. The culturing process may preferably be conducted underconditions in which the concentration of dissolved oxygen is between0.025 to 0.8 mol/m³, preferably 0.4 mol/m³. The oxygen level can becontrolled by any conventional means known to one skilled in the art,including but not limited to stirring and/or bubbling.

At the end of the culturing process, the P-decomposing yeast cells maybe recovered from the culture by various methods known in the art, andstored at a temperature below about 0° C. to 4° C. The P-decomposingyeast cells may also be dried and stored in powder form.

The amount of biologically available phosphorus, such as H₃PO₄, H₂PO₄ ⁻,and HPO₄ ²⁻, in the culture can be determined by any methods known inthe art, including but not limited to UV absorption spectroscopy. Theincrease can be calculated by the difference between the total amount ofbiologically available phosphorus in a culture with activated yeasts andthe amount of biologically available phosphorus in the same medium withnon-activated yeast. For example, 1 ml of Saccharomyces cerevisiaestrain AS2.399 (2 to 5×10⁷ yeasts/ml) is inoculated into 1000 ml of amedium according to Table 2. The culture is incubated at a temperatureof 28° C. in the presence of a series of 8 EM fields in the orderstated: 360 MHz at 14 mV/cm for 5 hours; 365 MHz at 14 mV/cm for 5hours; 370 MHz at 14 mV/cm for 5 hours; 380 MHz at 14 mV/cm for 5 hours;360 MHz at 130 mV/cm for 30 hours; 365 MHz at 130 mV/cm for 30 hours;370 MHz at 130 mV/cm for 30 hours; 375 MHz at 130 mV/cm for 30 hours.The increase in the amount of biologically available phosphorus wasdetermined to be greater than 330 mg/ml of yeast culture.

5.3. Phosphorus-Balancing Yeast Cell Component

The phosphorus-balancing (P-balancing) yeasts of the invention alsoconvert insoluble or biologically unavailable phosphorus-containingsubstances into soluble biologically available phosphorous compounds.However, the P-balancing yeast is preferably used when the level ofphosphorus in the local environment is high The conversion of insolubleor biologically unavailable phosphorus-containing substances intosoluble biologically available phosphorous is sensitive to the level ofphosphorus; at about 180 ppm or higher, the conversion is reduced whileat about 60 ppm or lower, the conversion is increased.

In the present invention, the P-balancing yeast cells are preferablydeployed in biologically fertilizer compositions that include an organicsubstrate that already contains a relatively significant level ofsoluble or biologically available phosphorous. For example, sludgecontains a relatively high level of soluble phosphorus as compared toother kinds of manure.

According to the invention, yeast cells that are capable of P-balancingare prepared by culturing the cells in the presence of anelectromagnetic field in an appropriate. culture medium. The frequencyof the electromagnetic field for activating or enhancing P-balancingfunction in yeasts can generally be found in the range of 300 MHz to 500MHz. After the cells have been cultured for a sufficient period of time,the cells can be tested for their ability to decomposephosphorus-containing substances by methods well known in the art.

The method of the invention for making the P-balancing yeast cells iscarried out in a liquid medium. The medium contains sources of nutrientsassimilable by the yeast cells. In general, carbohydrates such assugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 1.5%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

Among the inorganic salts which can be incorporated in the culture mediaare the customary salts capable of yielding sodium, potassium, calcium,sulfate, carbonate, and like ions. Non-limiting examples of nutrientinorganic salts are CaCO₃, MgSO₄, NaCl, and CaSO₄. Insolublephosphorus-containing substances in a suitable form are also included inthe media. Non-limiting examples include powder of dried sludge of >150mesh. Other insoluble phosphorus-containing substances can also be usedeither separately or in combination.

TABLE 3 Composition for a culture medium for P-balancing yeast MediumComposition Quantity Sucrose 15 g NaCl 1.2 g MgSO₄.7H₂O 0.2 g CaCO₃.5H₂O3.0 g CaSO₄.2H₂O 0.3 g KNO₃ 0.3 g Yeast extract paste 0.5 g Dried sludge1.2 g; Powder of > 150 mesh Autoclaved water 1000 ml

It should be noted that the composition of the media provided in Table 3is not intended to be limiting. Various modifications of the culturemedium may be made by those skilled in the art, in view of practical andeconomic considerations, such as the scale of culture and local supplyof media components.

The process can be initiated by inoculating 100 ml of medium with 1 mlof an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

The EM field(s), which can be applied by any means known in the art, caneach have a frequency in the range of about 300 to about 500 MHz, orpreferably in the range of 380.000 to 485.000 MHz. For example andwithout being limited by such examples, each EM field can have afrequency at about 380, 385, 390, 395, 400, 402, 405, 410, 415, 420,422, 425, 430, 432, 435, 440, 445, 450, 455, 460, 465, 470, 480 or 485MHz. The field strength of the EM field(s) is in the range of 90 to 300mV/cm. If a series of EM fields are applied, the EM fields can each havea different frequency within the stated range, or a different fieldstrength within the stated range, or different frequency and fieldstrength within the stated ranges. In a preferred embodiment, the EMfield(s) at the beginning of a series have a lower EM field strengththan later EM field(s), such that the yeast cell culture are exposed toEM fields of progressively increasing field strength. Although anypractical number of EM fields can be used within a series, it ispreferred that the yeast culture be exposed to a total of 2, 3, 4, 5, 6,7 or 8 different EM fields in a series.

Although the yeast cells will become activated even after a few hours ofculturing in the presence of the EM field(s), and the yeast cells can becultured in the presence of the EM field(s) for an extended period oftime (e.g., two or more weeks), it is generally preferred that theactivated yeast cells be allowed to multiply and grow in the presence ofthe EM field or EM fields for a total of about 230-480 hours.

For example, using an exemplary apparatus as depicted in FIG. 1, aninitial field strength in the range of 50-150 mV/cm, usually at about100 mV is used. After this first period of culture, the yeast cells arefurther incubated under substantially the same conditions for anotherperiod, except that the EM field strength is increased to a higher levelin the range of 200-300 mV/cm, usually to about 250 mV/cm. The processof the invention is carried out at temperatures ranging from about 23°to 30° C.; however, it is preferable to conduct the process at 25° to28° C. The culturing process may preferably be conducted underconditions in which the concentration of dissolved oxygen is between0.025 to 0.8 mol/m³, preferably 0.4 mol/m³. The oxygen level can becontrolled by any conventional means known to one skilled in the art,including but not limited to stirring and/or bubbling.

At the end of the culturing process, the P-balancing yeast cells may berecovered from the culture by various methods known in the art, andstored at a temperature below about 0° C. to 4° C. The P-balancing yeastcells may also be dried and stored in powder form.

The amount of biologically available phosphorus, such as H₃PO₄, H₂PO₄ ⁻,and HPO₄ ²⁻, in the culture can be determined by any methods known inthe art, including but not limited to UV absorption spectroscopy. Theincrease can be calculated by the difference between the total amount ofbiologically available phosphorus in a culture with activated yeasts andthe amount of phosphorus in the same medium with non-activated yeast.For example, 1 ml of Saccharomyces cerevisiae strain AS2.628 (2 to 5×10⁷yeasts/ml) is inoculated into 1000 ml of a medium containing 200 mg/l ofH₃PO₄, H₂PO₄ ⁻ and HPO₄ ²⁻. The culture is incubated at a temperature of28° C. in the presence of a series of 8 EM fields in the order stated:385 MHz at 99 mV/cm for 12 hours; 415 MHz at 99 mV/cm for 12 hours; 440MHz at 99 mV/cm for 12 hours; 460 MHz at 99 mV/cm for 12 hours; 385 MHzat 250 mV/cm for 48 hours; 415 MHz at 250 mV/cm for 48 hours; 440 MHz at250 mV/cm for 24 hours; 460 MHz at 250 mV/cm for 24 hours. The increasein the amount of biologically available phosphorus was determined to begreater than 24%. The control did not show any significant change in theamount of biologically as available phosphorus.

5.4. Potassium-Decomposing Yeast Cell Component

The potassium compound-decomposing (K-decomposing) yeasts of theinvention converts insoluble potassium-containing substances, such aspotassium mica, into soluble potassium so that they become available toplants.

In the present invention the ability of a plurality of yeast cells todecompose insoluble potassium-containing substances is activated orenhanced, and the resulting K-decomposing yeast cells can be used as acomponent of the biological fertilizer compositions of the invention.

According to the present invention, yeast cells that are capable ofK-decomposing are prepared by culturing the cells in the presence of anelectromagnetic field in an appropriate culture medium. The frequency ofthe electromagnetic field for activating or enhancing K-decomposition inyeasts can generally be found in the range of 100 MHz-300 MHz. After theyeast cells have been cultured for a sufficient period of time, thecells can be tested for their ability to decompose potassium-containingsubstances by methods well known in the art.

The method of the invention for making the K-decomposing yeast cells iscarried out in a liquid medium. The medium contains sources of nutrientsassimilable by the yeast cells. In general, carbohydrates such assugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 1.5%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

Among the inorganic salts which can be incorporated in the culture mediaare the customary salts capable of yielding sodium, calcium, phosphate,sulfate, carbonate, and like ions. Non-limiting examples of nutrientinorganic salts are (NH₄)₂HPO₄, CaCO₃, MgSO₄, NaCl, and CaSO₄. Insolublepotassium-containing substances in a suitable form are also included inthe media. Non-limiting examples include powder of potassium mica of≧200 mesh. Other insoluble potassium-containing substances can also beused either separately or combined.

TABLE 4 Composition for a culture medium for K-decomposing yeast MediumComposition Quantity Sucrose 15 g NaCl 1.2 g MgSO₄.7H₂O 0.2 g CaCO₃.5H₂O3.0 g CaSO₄.2H₂O 0.3 g (NH₄)₂HPO₄ 0.3 g Yeast extract paste 0.5 gPotassium mica 1.0 g, Powder of > 200 mesh Dried sludge 1.2-3 g, Powderof > 150 mesh Autoclaved water 1000 ml

It should be noted that the composition of the media provided in Table 4is not intended to be limiting. Various modifications of the culturemedium may be made by those skilled in the art, in view of practical andeconomic considerations, such as the scale of culture and local supplyof media components.

The process can be initiated by inoculating 100 ml of medium with 1 mlof an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

The EM field(s), which can be applied by any means known in the art, caneach have a frequency in the range of about 100 to about 300 MHz,preferably in the range of 190.000 to 285.000 MHz. For example andwithout being limited by such examples, each EM field can have afrequency at about 190, 195, 200, 205, 210, 215, 220, 225, 230, 235,240, 245, 250, 255, 260, 265, 270, 275, 280, or 285 MHz. The fieldstrength of the EM field(s) is in the range of 10 to 200 mV/cm. If aseries of EM fields are applied, the EM fields can each have a differentfrequency within the stated range, or a different field strength withinthe stated range, or different frequency and field strength within thestated ranges. In a preferred embodiment, the EM field(s) at thebeginning of a series have a lower EM field strength than later EMfield(s), such that the yeast cell culture are exposed to EM fields ofprogressively increasing field strength. Although any practical numberof EM fields can be used within a series, it is preferred that the yeastculture be exposed to a total of 2, 3, 4, 5, 6, 7, or 8 different EMfields in a series.

Although the yeast cells will become activated even after a few hours ofculturing in the presence of the EM field(s), and the yeast cells can becultured in the presence of the EM field(s) for an extended period oftime (e.g., one or more weeks), it is generally preferred that theactivated yeast cells be allowed to multiply and grow in the presence ofthe EM field or EM fields for a total of about 140-280 hours.

For example, using an exemplary apparatus as depicted in FIG. 1, aninitial field strength in the range of 10-20 mV/cm, usually at about 125mV/cm is used. After this first period of culture, the yeast cells arefurther incubated under substantially the same conditions for anotherperiod, except that the EM field strength is increased to a higher levelin the range of 50-200 mV/cm, usually to about 125 mV/cm. The process ofthe invention is carried out at temperatures ranging from about 23° to30° C.; however, it is preferable to conduct the process at 25° to 28°C. The culturing process may preferably be conducted under conditions inwhich the concentration of dissolved oxygen is between 0.025 to 0.8mol/m³, preferably 0.4 mol/m³. The oxygen level can be controlled by anyconventional means known to one skilled in the art, including but notlimited to stirring and/or bubbling.

At the end of the culturing process, the K-decomposing yeast cells maybe recovered from the culture by various methods known in the art, andstored at a temperature below about 0-4° C. The K-decomposing yeastcells may also be dried and stored in powder form.

Any methods known in the art can be used to test the cultured yeastcells for their ability to decompose insoluble potassium-containingsubstances. For example, 1 ml of Saccharomyces cerevisiae strain AS2.631(2 to 5×10⁷ cells/ml) was inoculated into 1000 ml of a medium accordingto Table 4. The culture was incubated at a temperature at 28° C. in thepresence of a series of 8 EM fields in the order stated: 210 MHz at 14mV/cm for 5 hours; 235 MHz at 14 mV/cm for 5 hours; 245 MHz at 14 mV/cmfor 5 hours; 255 MHz at 14 mV/cm for 5 hours; 210 MHz at 120 mV/cm for30 hours; 235 MHz at 120 mV/cm for 30 hours; 245 MHz at 120 mV/cm for 30hours; 255 MHz at 120 mV/cm for 30 hours. A control was set up whichcontained non-activated cells of the same strain of yeasts. The amountof biologically available potassium K⁺ in the culture can be determinedby any methods known in the art, including but not limited to flamespectroscopy and/or atomic absorption spectrometry. The increase inpotassium is calculated by the difference between the quantity ofpotassium in the medium of Table 4 after culturing and the basal levelof potassium in the medium prior to culturing. The increase in theamount of biologically available potassium was determined to be greaterthan 120 mg/ml of cultured yeast cells. There was no significant changein the amount of potassium available in the control.

5.5. Complex Carbon-Decomposing Yeast Cell Component

The carbon-decomposing (C-decomposing) yeast of the invention convertscomplex, high molecular weight, carbon compounds and materials, inparticular, complex carbohydrates, such as cellulose and lignin, intosimple carbohydrates, such as pentoses and hexoses. Such simplecarbohydrates are utilized by other yeast cells in the compositions tosupport their growth and activities.

In a preferred embodiment, yeast cells are used to make theC-decomposing yeast cell component of the invention. In the presentinventions the ability of yeast to decompose complex carbon compoundsefficiently is activated or enhanced, and the resulting C-decomposingyeast cells can be used as a component of the biological fertilizercomposition of the invention.

According to the present invention, yeast cells that are capable ofC-decomposition are prepared by culturing the cells in the presence ofan electromagnetic field in an appropriate culture medium. The frequencyof the electromagnetic field for C-decomposition in yeasts can generallybe found in the range of 1000 MHz-1200 MHz. After the yeast cells havebeen cultured for a sufficient period of time, the cells can be testedfor their ability to decompose complex carbon compounds by methods wellknown in the art.

The method of the invention for making the C-decomposing yeast cells iscarried out in a liquid medium. The medium contains sources of nutrientsassimilable by the yeast cells. Complex carbon-containing substancessuch as cellulose, lignin, coal powder, etc., in a suitable form can beused as sources of carbon in the culture medium. The exact quantity ofthe carbon source or sources utilized in the medium depends in part uponthe other ingredients of the medium but, in general, the amount ofsimple carbohydrate usually varies between about 0.1% and 5% by weightof the medium and preferably between about 0.1% and 1%, and mostpreferably about 0.5%. These carbon sources can be used individually, orseveral such carbon sources may be combined in the medium.

Among the inorganic salts which can be incorporated in the culture mediaare the customary salts capable of yielding sodium, calcium, phosphate,sulfate, carbonate, and like ions. Non-limiting examples of nutrientinorganic salts are (NH₄)₂HPO₄, K₂HPO₄, CaCO₃, MgSO₄, NaCl, and CaSO₄.

TABLE 5 Composition for a culture medium for C-decomposing yeasts MediumComposition Quantity Cellulose 3.0 g; Powder of > 100 mesh Dried sludge5 g; Powder of > 150 mesh NaCl 0.6 g MgSO₄.7H₂O 0.3 g CaCO₃.5H₂O 1.5 gCaSO₄.2H₂O 0.4 g (NH₄)₂HPO₄ 0.3 g Yeast extract paste 0.5 g K₂HPO₄ 0.5 gAutoclaved water 1000 ml

It should be noted that the composition of the media provided in Table 5is not intended to be limiting. Various modifications of the culturemedium may be made by those skilled in the art, in view of practical andeconomic considerations, such as the scale of culture and local supplyof media components.

The process can be initiated by inoculating 100 ml of medium with 1 mlof an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

The EM field(s), which can be applied by any means known in the art, caneach have a frequency in the range of about 1000 to about 1200 MHz,preferably in the range of 1050.000 to 1160.000 MHz. For example andwithout being limited by such examples, each EM field can have afrequency at about 1050, 1055, 1060, 1065, 1070, 1075, 1080, 1085, 1090,1095, 1100, 1105, 1110, 1115, 1120, 1125, 1130, 1135, 1140, 1145, 1150,1155, or 1160 MHz. The field strength of the EM field(s) is in the rangeof 10 to 200 mV/cm. If a series of EM fields are applied, the EM fieldscan each have a different frequency within the stated range, or adifferent field strength within the stated range, or different frequencyand field strength within the slated ranges. In a preferred embodiment,the EM field(s) at the beginning of a series have a lower EM fieldstrength than later EM field(s), such that the yeast cell culture areexposed to EM fields, of progressively increasing field strength.Although any practical number of EM fields can be used within a series,it is preferred that the yeast culture be exposed to a total of 2, 3, 4,5, 6, 7, or 8 different EM fields in a series.

Although the yeast cells will become activated even after a few hours ofculturing in the presence of the EM field(s), and the yeast cells can becultured in the presence of the EM field(s) for an extended period oftime (e.g., one or more weeks), it is generally preferred that theactivated yeast cells be allowed to multiply and grow in the presence ofthe EM field or EM fields for a total of about 140-280 hours.

For example, using an exemplary apparatus as depicted in FIG. 1, aninitial field strength in the range of 10-20 mV, usually at about 12.5mV/cm is used. After this first period of culture, the yeast cells arefurther incubated under substantially the same conditions for anotherperiod, except that the EM field strength is increased to a higher levelin the range of 100-200 mV/cm, usually to about 125 mV/cm. The processof the invention is carried out at temperatures ranging from about 23°to 30° C.; however, it is preferable to conduct the process at 25° to28° C. The culturing process may preferably be conducted underconditions in which the concentration of dissolved oxygen is between0.025 to 0.8 mol/m³, preferably 0.4 mol/m³. The oxygen level can becontrolled by any conventional means known to one skilled in the art,including but not limited to stirring and/or bubbling.

At the end of the culturing process, the C-decomposing yeast cells maybe recovered from the culture by various methods known in the art, andstored at a temperature below about 0-4° C. The C-decomposing yeastcells may also be dried and stored in powder form.

Any methods known in the art can be used to test the cultured yeastcells for their ability to decompose complex-carbon containingsubstances. For example, a change in the chemical oxygen demand (COD) ofa sample can be used as an indication of the change in the concentrationof complex-carbon containing substances in the sample. For example, 1 mlof the Saccharomyces cerevisiae strain AS2.982 (2 to 5×10⁷ yeasts/ml) isinoculated into 30 ml of a medium according to Table 5. The culture isincubated at a temperature in the range of 20-28° C. for in the presenceof a series of 8 EM fields in the order stated: 1050 MHz at 16 mV/cm for5 hours; 1070 MHz at 16 mV/cm for 5 hours; 1090 MHz at 16 mV/cm for 5hours; 1110 MHz at 16 mV/cm for 5 hours; 1,050 MHz at 125 mV/cm for 30hours; 1070 MHz at 125 mV/cm for 30 hours; 1090 MHz at 125 mV/cm for 30hours; 1110 MHz at 125 mV/cm for 30 hours. After activation, based on achange in COD, the amount of carbohydrates in the culture was estimatedto be greater than 330 mg/ml of yeast culture.

Alternatively, the amount of simple carbohydrates in the culture canthen he determined by any methods known in the art including but notlimited to biochemical reactions, chromatography and molecularfluorescence spectroscopy.

5.6. Growth Factors Producing Yeast Cell Component

The growth factors producing (GF-producing) yeast of the presentinvention produces many vitamins and other nutrients, such as but notlimited to, vitamin B-1, riboflavin (vitamin B-2), vitamin B-12, niacin(B-3), pyridoxine (B-6), pantothenic acid (B-5), folic acid, biotin,para-aminobenzoic acid, choline, inositol, in such amounts that cansupport the growth of other yeast strains.

The ability of yeast to overproduce growth factors is activated orenhanced by methods of this invention, and the resulting GF-producingyeast cells are included as a component of the biological fertilizercomposition of the invention.

According to the present invention, yeast cells that are capable ofoverproducing growth factors are prepared by culturing the yeast cellsin the presence of an electromagnetic field in an appropriate culturemedium. The frequency of the electromagnetic field for activating orenhancing GF-production in yeasts can generally be found in the range of1300 MHz-1500 MHz. After the yeast cells have been cultured for asufficient period of time, the cells can be tested for their ability toproduce growth factors by methods well known in the art.

The method of the invention for making the GF-producing yeast cells iscarried out in a liquid medium. The medium contains sources of nutrientsassimilable by the yeast cells. In general, carbohydrates such assugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 0.8%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

Among the inorganic salts which can be incorporated in the culture mediaare the customary salts capable of yielding sodium, calcium, phosphate,sulfate carbonate, and like ions. Non-limiting examples of nutrientinorganic salts are NH₄NO₃, K₂HPO₄, CaCO₃, MgSO₄, NaCl, and CaSO₄.

TABLE 6 Composition for a culture medium for GF-producing yeasts MediumComposition Quantity Starch 8.0 g; Powder of > 120 mesh NaCl 0.3 gMgSO₄.7H₂O 0.2 g CaCO₃.5H₂O 0.5 g CaSO₄.2H₂O 0.2 g NH₄NO₃ 0.3 g K₂HPO₄0.8 g Autoclaved water 1000 ml

It should be noted that the composition of the media provided in Table 6is not intended to be limiting. Various modifications of the culturemedium may be made by those skilled in the art, in view of practical andeconomic considerations, such as the scale of culture and local supplyof media components.

The process can be initiated by inoculating 100 ml of medium with 1 mlof an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

The EM field(s), which can be applied by any means known in the art, caneach have a frequency in the range of about 1300 to about 1500 MHz,preferably in the range of 1340.000 to 1440.000 MHz. For example andwithout being limited by such examples, each EM field can have afrequency at about 1340, 1345, 1350, 1355, 1360, 1365, 1370, 1375, 1380,1385, 1390, 1395, 1400, 1405, 1410, 1415, 1420, 1425, 1430, 1435, or1440 MHz. The field strength of the EM field(s) is in the range of 20 to200 mV/cm. If a series of EM fields are applied, the EM fields can eachhave a different frequency within the stated range, or a different fieldstrength within the stated range, or different frequency and fieldstrength within the stated ranges. In a preferred embodiment, the EMfield(s) at the beginning of a series have a lower EM field strengththan later EM field(s), such that the yeast cell culture are exposed toEM fields of progressively increasing field strength. Although anypractical number of EM fields can be used within a series, it ispreferred that the yeast culture be exposed to a total of 2, 3, 4, 5, 6,7 or 8 different EM fields in a series.

Although the yeast cells will become activated even after a few hours ofculturing in the presence of the EM field(s), and the yeast cells can becultured in the presence of the EM field(s) for an extended period oftime (e.g., one or more weeks), it is generally preferred that theactivated yeast cells be allowed to multiply and grow in the presence ofthe EM field or EM fields for a total of about 140-280 hours.

For example, using an exemplary apparatus as depicted in FIG. 1, aninitial field strength in the range of 20-40 mV/cm, usually at about 25mV/cm is used. After this first period of culture, the yeast cells arefurther incubated under substantially the same conditions for anotherperiod, except that the amplitude is increased to a higher level in therange of 100-200 mV/cm, usually to about 125 mV. The process of theinvention is carried out at temperatures ranging from about 23° to 30°C.; however, it is preferable to conduct the process at 25° to 28° C.The culturing process may preferably be conducted under conditions inwhich the concentration of dissolved oxygen is between 0.025 to 0.8mol/m³, preferably 0.4 mol/m³. The oxygen level can be controlled by anyconventional means known to one skilled in the art, including but notlimited to stirring and/or bubbling.

At the end of the culturing process, the GF-producing yeast cells may berecovered from the culture by various methods known in the art, andstored at a temperature below about 0-4° C. The GF-producing yeast cellsmay also be dried and stored in powder form.

Any methods known in the art can be used to test the cultured yeastcells for their ability to overproduce growth factors, including but notlimited to high performance liquid chromatography (HPLC). For example, 1ml of activated or non-activated Saccharomyces cerevisiae strain AS2.413(2 to 5×10⁷ yeasts/ml) was inoculated into 1000 ml of a medium accordingto Table 6. The culture was incubated at a temperature of 28° C. in thepresence of a series of 8 EM fields in the order stated: 1340 MHz at 28mV/cm for 5 hours; 1350 MHz at 28 mV/cm for 5 hours; 1380 MHz at 28mV/cm for 5 hours; 1390 MHz at 28 mV/cm for 5 hours; 1340 MHz at 135mV/cm for 30 hours; 1350 MHz at 135 mV/cm for 30 hours; 1380 MHz at 135mV/cm for 30 hours; 1390 MHz at 135 mV/cm for 30 hours. The amount ofgrowth factors produced can be calculated by the difference between thetotal amount of vitamin B1, B2, B6, B12 in a culture with activated ornon-activated yeasts and the total amount of the same growth factors inthe same medium without yeast. The increase in the amount of growthfactors was determined to be greater than 350 mg/ml of activated yeastculture.

5.7. ATP-Producing Yeast Cell Component

The ATP-producing yeast of the present invention is capable ofoverproducing ATP in such amounts that can support the growth of othermicrobes in the biological fertilizer compositions.

In the present invention, the ability of yeast to overproduce ATP isactivated or enhanced, and the resulting ATP-producing yeast cells canbe used as a component of the biological fertilizer compositions of theinvention.

According to the present invention, yeast cells that are capable ofenhanced ATP-production are prepared by culturing the cells in thepresence of an electric field in an appropriate culture medium. Thefrequency of the electromagnetic field for activating or enhancingATP-production in yeasts can generally be found in the range of 1600MHz-1800 MHz. After sufficient time is given for the cells to grow, thecells can be tested for their enhanced ability to produce ATP by methodswell known in the art.

The method of the invention for making the ATP-producing yeast cells iscarried out in a liquid medium. The medium contains sources of nutrientsassimilable by the yeast cells. In general, carbohydrates such assugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 0.8%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

Among the inorganic salts which can be incorporated in the culture mediaare the customary salts capable of yielding sodium, calcium, phosphate,sulfate, carbonate, and like ions. Non-limiting examples of nutrientinorganic salts are (NH₄)₂HPO₄, K₂HPO₄, CaCO₃, MgSO₄, NaCl, and CaSO₄.

TABLE 7 Composition for a culture medium for ATP-producing yeasts MediumComposition Quantity Starch 10.0 g, 120 > mesh NaCl 0.2 g MgSO₄.7H₂O 0.2g CaCO₃.5H₂O 0.8 g CaSO₄.2H₂O 0.2 g NH₄NO₃ 0.2 g K₂HPO₄ 0.5 g Autoclavedwater 1000 ml

It should be noted that the composition of the media provided in Table 7is not intended to be limiting. Various modifications of the culturemedium may be made by those skilled in the art, in view of practical andeconomic considerations, such as the scale of culture and local supplyof media components.

The process can be initiated by inoculating 100 ml of medium with 1 mlof an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

The EM field(s), which can be applied by any means known in the art, caneach have a frequency in the range of about 1600 to about 1800 MHz,preferably in the range of 1630.000 to 1730.000 MHz. For example andwithout being limited by such examples, each EM field can have afrequency at about 1630, 1635, 1640, 1645, 1650, 1655, 1660, 1665, 1670,1675, 1680, 1685, 1690, 1695, 1700, 1705, 1710, 1715, 1720, 1725, or1730 MHz. The field strength of the EM field(s) is in the range of 20 to200 mV/cm. If a series of EM fields are applied, the EM fields can eachhave a different frequency within the stated range, or a different fieldstrength within the stated range, or different frequency and fieldstrength within the stated ranges. In a preferred embodiment, the EMfield(s) at the beginning of a series have a lower EM field strengththan later EM field(s), such that the yeast cell culture are exposed toEM fields of progressively increasing field strength. Although anypractical number of EM fields can be used within a series, it ispreferred that the yeast culture be exposed to a total of 2, 3, 4, 5, 6,7, or 8 different EM fields in a series.

Although the yeast cells will become activated even after a few hours ofculturing in the presence of the EM field(s), and the yeast cells can becultured in the presence of the EM field(s) for an extended period ortime (e.g., one or more weeks), it is generally preferred that theactivated yeast cells be allowed to multiply and grow in the presence ofthe EM field or EM fields for a total of about 160-300 hours.

For example, using an exemplary apparatus as depicted in FIG. 1, aninitial field strength in the range of 20-40 mV/cm, usually at about 30mV/cm is used. After this first period of culture, the yeast cells arefurther incubated under substantially the same conditions for anotherperiod, except that the amplitude is increased to a higher level in therange of 100-200 mV/cm, usually to about 150 mV/cm. The process of theinvention is carried out at temperatures ranging from about 23° to 30°C.; however, it is preferable to conduct the process at 25° to 28° C.The culturing process may preferably be conducted under conditions inwhich the concentration of dissolved oxygen is between 0.025 to 0.8mol/m³, preferably 0.4 mol/m³. The oxygen level can be controlled by anyconventional means known to one skilled in the art, including but notlimited to stirring and/or bubbling.

At the end of the culturing process, the ATP-producing yeast cells maybe recovered from the culture by various methods known in the art, andstored at a temperature below about 0-4° C. The ATP-producing yeastcells may also be dried and stored in powder form.

Any methods known in the art can be used to test the cultured yeastcells for their ability to overproduce ATP, including but not limited toHPLC. For example, 1 ml of the activated yeast culture (2 to 5×10⁷yeasts/ml) was inoculated into 1000 ml of a medium according to Table 7.The culture was incubated at a temperature of 28° C. in the presence ofa series of 8 EM fields in the order stated: 1635 MHz at 29 mV/cm for 10hours; 1655 MHz at 29 mV/cm for 10 hours; 1675 MHz at 29 mV/cm for 10hours; 1695 MHz at 29 mV/cm for 10 hours; 1635 MHz at 150 mV/cm for 30hours; 1655 MHz at 150 mV/cm for 30 hours; 1675 MHz at 150 mV/cm for 30hours; 1695 MHz at 150 mV/cm for 30 hours. The amount of ATP producedcan be calculated by the difference between the total amount of ATP in aculture with yeasts and the amount of ATP in the same medium withoutyeast. Using activated Saccharomyces cerevisiae strain AS2.536, theamount of ATP in the culture was determined to be 170 mg/ml of yeastculture.

5.8 Pathogen-Suppressing Yeast Cell Component

The present invention also provides yeast cells that are capable ofsuppressing the proliferation of pathogenic microorganisms that arepresent in the materials used in the organic substrate component of thebiological fertilizer Typically, due to an abundance of nutrientspresent in the organic substrate material for such pathogenicmicroorganisms, the numbers of pathogens increase rapidly over a periodof time. However, in the presence of the pathogen-suppressing yeasts ofthe invention, the numbers of pathogens in the organic substratematerial remains unchanged, or decreases over time. Without being boundby any theory or mechanism, the inventor believes that the presence ofthe pathogen-suppressing yeasts in the organic substrate materialcreates an environment that is unfavorable for the growth of pathogenicmicroorganisms.

According to the invention, the ability of yeasts to affect/control thenumbers of pathogens is activated or enhanced by culturing the yeasts inthe presence of an electromagnetic field. The resultingpathogen-suppressing yeast cells are used as a component in thebiological fertilizer compositions of the invention.

The frequency of the electromagnetic field for activating or enhancingthe ability of yeasts to control the numbers of pathogenicmicroorganisms can generally be found in the range of 30 MHz to 50 MHz.After sufficient time is given for the yeast cells to grow, the cellscan be tested for their ability to affect/control the number ofpathogens by methods well known in the art.

The method of the invention for making pathogen-suppressing yeast cellsis carried out in a liquid medium. The medium contains sources ofnutrients assimilable by the yeast cells. In general, carbohydrates suchas sugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%.and most preferably about 0.8%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

Among the inorganic salts which can be incorporated in the culture mediaare the customary salts capable of yielding sodium, calcium, phosphate,sulfate, carbonate, and like ions. Non-limiting examples of nutrientinorganic salts are (NH₄)₂HPO₄, K₂HPO₄, CaCO₃, MgSO₄, NaCl, and CaSO₄.

TABLE 8 Composition for a culture medium for Pathogen-Suppressing yeastsMedium Composition Quantity Soluble Starch 8.0 g Sucrose 5 g NaCl 0.2 gMgSO₄.7H₂O 0.2 g CaCO₃.5H₂O 0.5 g CaSO₄.2H₂O 0.2 g Peptone 1.5 g K₂HPO₄0.5 g Autoclaved water 400 ml Sludge extract 600 ml

The sludge extract for the culture medium is prepared by incubating 500g of sludge in about 600 ml of warm water (at 35° C. to 40° C.) for 24hours at 30-37° C., and filtering the fluid to remove particulatematters. It should be noted that the composition of the media providedin Table 8 is not intended to be limiting. Various modifications of theculture medium may be made by those skilled in the art, in view ofpractical and economic considerations, such as the scale of culture andlocal supply of media components.

The process can be initiated by inoculating 100 ml of medium with 1 mlof an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

The EM field(s), which can be applied by any means known in the art, caneach have a frequency in the range of about 30.000 to about 50.000 MHz.For example and without being limited by such examples each EM field canhave a frequency at about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 MHz. The field strength of theEM field(s) is in the range of 0.5 to 200 mV/cm, preferably 10 to 180mV/cm. If a series of EM fields are applied, the EM fields can each havea different frequency within the stated range, or a different fieldstrength within the stated range or different frequency and fieldstrength within the stated ranges. In a preferred embodiment, the EMfield(s) at the beginning of a series have a lower EM field strengththan later EM field(s), such that the yeast cell culture are exposed toEM fields of progressively increasing field strength. Although anypractical number of EM fields can be used within a series, it ispreferred that the yeast culture be exposed to a total of 2, 3, 4, 5, 6,7, or 8 different EM fields in a series.

Although the yeast cells will become activated even after a few hours ofculturing in the presence of the EM field(s), and the yeast cells can becultured in the presence of the EM field(s) for an extended period oftime (e.g., one or more weeks), it is generally preferred that theactivated yeast cells be allowed to multiply and grow in the presence ofthe EM field or EM fields for a total of about 144-272 hours.

For example, using an exemplary apparatus as depicted in FIG. 1, aninitial field strength in the range of 10-30 mV/cm, usually at about 25mV/cm is used. After this first period of culture, the yeast cells arefurther incubated under substantially the same conditions for anotherperiod, except that the amplitude is increased to a higher level in therange of 100-200 mV/cm, usually to about 150 mV/cm. The process of theinvention is carried out at temperatures ranging from about 23° to 30°C.; however, it is preferable to conduct the process at 25° to 28° C.The culturing process may preferably be conducted under conditions inwhich the concentration of dissolved oxygen is between 0.025 to 0.8mol/m³, preferably 0.4 mol/m³. The oxygen level can be controlled by anyconventional means known to one skilled in the art, including but notlimited to stirring and/or bubbling.

At the end of the culturing process, the pathogen-suppressing yeastcells may be recovered from the culture by various methods known in theart, and stored at about 0° C. to 4° C. The pathogen-suppressing yeastcells may also be dried and stored in powder form.

The ability of the pathogen-suppressing yeasts to control the numbers ofpathogens can be determined by any methods known in the art forenumerating microorganisms, such as optical density, plating outdilutions on solid media for counting, or counting individual cellsunder a microscope. Stains may be applied to distinguish or identifydifferent strains or species of microorganisms present in a sample, orto determine their viability. When a range of pathogenic microorganismsare expected to be affected by the pathogen-suppressing yeasts, thenumbers of move than one representative species of pathogenicmicroorganisms can be monitored to assess the performance of thepathogen-suppressing yeasts.

For example, samples of organic substrate material containing a knownconcentration of pathogenic microorganisms are cultured under the sameconditions for a same period of time in the presence of differentconcentrations of pathogen-suppressing yeasts, and as negative control,the same strain of yeasts that have not been treated according to theculturing methods of the invention. A sample without any added yeast mayalso be included to determine the growth of pathogens under normalcircumstances. The numbers of pathogens before and after the cultureperiod are determined and compared.

A one liter culture containing at least 10¹⁰ cells of a pathogenicmicroorganism per ml is prepared. One ml of activated yeast cells(containing 2 to 5×10⁷ yeasts per ml) is added to the one liter cultureof pathogenic microorganism and incubated at 30° C. for 24 hours.Controls are included which contained non-activated yeast cells or noyeasts. The numbers of microorganisms in the respective cultures arethen determined and compared. The following are several examples inwhich a particular species of pathogenic bacteria was studied.

Using cells of Saccharomyces cerevisiae strain IFFI1037 which had beencultured in the presence of a series of 8 EM fields in the order stated:30 MHz at 26 mV/cm for 12 hours; 36 MHz at 26 mV/cm for 12 hours; 43 MHzat 26 mV/cm for 12 hours; 47 MHz at 26 mV/cm for 12 hours; 30 MHz at 150mV/cm for 24 hours; 36 MHz at 150 mV/cm for 24 hours; 43 MHz at 150mV/cm for 24 hours; 47 MHz at 150 mV/cm for 24 hours. The number ofStaphylococcus aureus in a sample was reduced by more than 2.7% relativeto the control with no yeasts. There was no significant change in thenumber of pathogens in the control containing non-activated cells.

Using cells of Saccharomyces cerevisiae strain IFFI1021 which had beencultured in the presence of a series of 8 EM fields in the order stated:30 MHz at 26 mV/cm for 12 hours; 36 MHz at 26 mV/cm for 12 hours; 42 MHzat 26 mV/cm for 12 hours; 49 MHz at 26 mV/cm for 12 hours; 30 MHz at 150mV/cm for 24 hours; 36 MHz at 150 mV/cm for 24 hours; 42 MHz at 150mV/cm for 24 hours; 49 MHz at 150 mV/cm for 24 hours. The number ofDiplococcus pneumoniae in a sample was reduced by more than 2.8%relative to the control with no yeasts. There was no significant changein the number of pathogens in the control containing non-activatedcells.

Using cells of Saccharomyces cerevisiae strain IFFI1051 which had beencultured in the presence of a series of 8 EM fields in the order stated:35 MHz at 26 mV/cm for 12 hours; 39 MHz at 26 mV/cm for 12 hours; 43 MHzat 26 mV/cm for 12 hours; 47 MHz at 26 mV/cm for 12 hours; 35 MHz at 150mV/cm for 24 hours; 39 MHz at 150 mV/cm for 24 hours; 43 MHz at 150mV/cm for 24 hours; 47 MHz at 150 mV/cm for 24 hours. The number ofBacillus anthracis in a sample was reduced by, more than 3.1% relativeto the control with no yeasts. There was no significant change in thenumber of pathogens in the control containing non-activated cells.

Using cells of Saccharomyces cerevisiae strain IFFI1331 which had beencultured in the presence of a series of 8 EM fields in the order,stated: 33 MHz at 26 mV/cm for 12 hours; 36 MHz at 26 mV/cm for 12hours; 45 MHz at 26 mV/cm for 12 hours; 47 MHz at 26 mV/cm for 12 hours;33 MHz at 150 mV/cm for 24 hours; 36 MHz at 150 mV/cm for 24 hours; 45MHz at 150 mV/cm for 24 hours; 47 MHz at 150 mV/cm for 24 hours. Thenumber of Mycobacterium tuberculosis in a sample was reduced by morethan 2.9% relative to the control with no yeasts. There was nosignificant change in the number of pathogens in the control containingnon-activated cells.

Using cells of Saccharomyces cerevisiae strain IFFI1345 which had beencultured in the presence of a series of 8 EM fields in the order stated:30 MHz at 26 mV/cm for 12 hours; 34 MHz at 26 mV/cm for 12 hours; 38 MHzat 26 mV/cm for 12 hours; 49 MHz at 26 mV/cm for 12 hours; 30 MHz at 150mV/cm for 24 hours; 34 MHz at 150 mV/cm for 24 hours; 38 MHz at 150mV/cm for 24 hours; 49 MHz at 150 mV/cm for 24 hours. The number ofEscherichia coli in a sample was reduced by more than 48% relative tothe control with no yeasts. There was no significant change in thenumber of pathogens in the control containing non-activated cells.

Using cells of Saccharomyces cerevisiae strain IFFI1211 which had beencultured in the presence of a series of 8 EM fields in the order stated:30 MHz at 26 mV/cm for 12 hours; 33 MHz at 26 mV/cm for 12 hours; 36 MHzat 26 mV/cm for 12 hours; 38 MHz at 26 mV/cm for 12 hours; 30 MHz at 150mV/cm for 24 hours; 33 MHz at 150 mV/cm for 24 hours; 36 MHz at 150mV/cm for 24 hours; 38 MHz at 150 mV/cm for 24 hours. The number ofSalmonella species bacteria in a sample was reduced by more than 66%relative to the control with no yeasts. There was no significant changein the number of pathogens in the control containing non-activatedcells.

5.9. Yeast Cell Component that Decomposes Undesirable Chemicals

The present invention further provides yeast cells that are capable ofdegrading undesirable chemicals, such as antibiotics, that are typicallyfound in manure

According to the invention, the ability of yeasts to degrade antibioticsis activated or enhanced by culturing the yeasts in the presence of anelectromagnetic field. The resulting yeast cells can be used as acomponent in the biological fertilizer compositions of the invention.

The frequency of the electromagnetic field for activating or enhancingthe ability of yeasts to degrade undesirable chemicals, in particularantibiotics, can generally be found in the range of 70 MHz to 100 MHz.After sufficient time is given for the yeast cells to grow, the yeastcells can be tested for their enhanced ability to decompose antibioticsby methods well known in the art. Antibiotics degraded by the yeasts ofthe invention include but are not limited to molecules within thefamilies of beta-lactams, tetracyclines, polypeptides, glycopeptides,aminoglycosides, and macrolides.

The method of the invention for making antibiotics-degrading yeasts iscarried out in a liquid medium. The medium contains sources of nutrientsassimilable by the yeast cells. In general, carbohydrates such assugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 0.8%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

Among the inorganic salts which can be incorporated in the culture mediaare the customary salts capable of yielding sodium, calcium, phosphate,sulfate, carbonate, and like ions. Non-limiting examples of nutrientinorganic salts are (NH₄)₂HPO₄, K₂HPO₄, CaCO₃, MgSO₄, NaCl, and CaSO₄.

TABLE 9 Composition for a culture medium for yeasts that degradeundesirable chemicals Medium Composition Quantity Soluble Starch 8.0g, > 120 mesh Sucrose 5 g NaCl 0.2 g MgSO₄.7H₂O 0.2 g CaCO₃.5H₂O 0.5 gCaSO₄.2H₂O 0.2 g Peptone 15 g K₂HPO₄ 0.5 g Autoclaved water 1000 mlSludge extract 600 ml

The sludge extract for the culture medium is prepared by incubating 500g of fresh sludge in about 600 ml of warm water (at 35-40° C.) for 24hours at 30-37° C., and filtering the fluid to remove particulatematters. It should be noted that the composition of the media providedin Table 9 is not intended to be limiting. Various modifications of theculture medium may be made by those skilled in the art, in view ofpractical and economic considerations, such as the scale of culture andlocal supply of media components.

The process can be initiated by inoculating 100 ml of medium with 1 mlof an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

The EM field(s), which can be applied by any means known in the art, caneach have a frequency in the range of 70.000 to 100.000 MHz. For exampleand without being limited by such examples, each EM field can have afrequency at about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 100MHz. The field strength of the EM field(s) is in the range of 40 to 250mV/cm. If a series of EM fields are applied, the EM fields can each havea different frequency within the stated range, or a different fieldstrength within the stated range, or different frequency and fieldstrength within the stated ranges. In a preferred embodiment, the EMfield(s) at the beginning of a series have a lower EM field strengththan later EM field(s), such that the yeast cell culture are exposed toEM fields of progressively increasing field strength. Although anypractical number of EM fields can be used within a series, it ispreferred that the yeast culture be exposed to a total of 2, 3, 4, 5, 6,7, or 8 different EM fields in a series.

Although the yeast cells will become activated even after a few hours ofculturing in the presence of the EM field(s), and the yeast cells can becultured in the presence of the EM field(s) for an extended period oftime (e.g., one or more weeks), it is generally preferred that theactivated yeast cells be allowed to multiply and grow in the presence ofthe EMs field or EM fields for a total of about 180-328 hours.

For example, using an exemplary apparatus as depicted in FIG. 1, aninitial field strength in the range of 40-60 mV/cm, usually at about 50mV is used. After this first period of culture, the yeast cells arefurther incubated under substantially the same conditions for anotherperiod, except that the amplitude is increased to a higher level in therange of 100-250 mV/cm, usually to about 200 mV/cm. The process of theinvention is carried out at temperatures ranging from about 23° to 30°C.; however, it is preferable to conduct the process at 25° to 28° C.The culturing process may preferably be conducted under conditions inwhich the concentration of dissolved oxygen is between 0.025 to 0.8mol/m³, preferably 0.4 mol/m³. The oxygen level can be controlled by anyconventional means known to one skilled in the art, including but notlimited to stirring and/or bubbling.

At the end of the culturing process, the yeast cells may be recoveredfrom the culture by various methods known in the art, and stored at atemperature below about 0° C. to 4° C. The recovered yeast cells mayalso be dried and stored in powder.

To determine the activity of the activated yeast cells towards anantibiotic compound, methods well known in the art, such as HPLC, can beused to measure the amounts of the antibiotic compound in a test sampleat various time point and under different incubation conditions. Forexample, a known amount of an antibiotic (up to 100 mg per liter) isadded to 10 liter of an aqueous extract of the manure. Then, 0.1 ml eachof activated and non-activated yeasts (at least 10⁷ cells/ml) are addedto the 10 liter samples containing the antibiotics, and incubated for 24hours at 28° C. A control is also included which does not contain anyyeast cells. After 24 hours, the amounts of antibiotics remaining in theextracts are determined and compared by performing HPLC on samples ofthe extracts.

Using cells of Saccharomyces cerevisiae strain AS2.293 which had beencultured in the presence of a series of 8 EM fields in the order stated:77 MHz at 48 mV/cm for 15 hours; 83 MHz at 48 mV/cm for 15 hours; 90 MHzat 48 mV/cm for 15 hours; 96 MHz at 48 mV/cm for 15 hours; 77 MHz at 200mV/cm for 30 hours; 83 MHz at 200 mV/cm for 30 hours; 90 MHz at 200mV/cm for 30 hours; 96 MHz at 200 mV/cm for 30 hours. The amount ofpenicillin G in a sample was reduced by more than 23% relative to thecontrol with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

Using cells of Saccharomyces cerevisiae strain IFFI1063 which had beencultured in the presence of a series of 8 EM fields in the order stated:70 MHz at 48 mV/cm for 15 hours; 73 MHz at 48 mV/cm for 15 hours; 88 MHzat 48 mV/cm for 15 hours; 98 MHz at 48 mV/cm for 15 hours; 70 MHz at 200mV/cm for 30 hours; 73 MHz at 200 mV/cm for 30 hours; 88 MHz at 200mV/cm for 30 hours; 98 MHz at 200 mV/cm for 30 hours. The amount ofchlorotetracycline in a sample was reduced by more than 31% relative tothe control with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

Using cells of Saccharomyces cerevisiae strain IFFI1221 which had beencultured in the presence of a series of 8 EM fields in the order stated:70 MHz at 48 mV/cm for 15 hours; 74 MHz at 48 mV/cm for 15 hours; 88 MHzat 48 mV/cm for 15 hours; 98 MHz at 48 mV/cm for 15 hours; 70 MHz at 200mV/cm for 30 hours; 74 MHz at 200 mV/cm for 30 hours; 88 MHz at 200mV/cm for 30 hours; 98 MHz at 200 mV/cm for 30 hours. The amount ofoxytetracycline in a sample was reduced by more than 28% relative to thecontrol with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

Using cells of Saccharomyces cerevisiae strain IFFI1340 which had beencultured in the presence of a series of 8 EM fields in the order stated:71 MHz at 48 mV/cm for 15 hours; 73 MHz at 48 mV/cm for 15 hours; 77 MHzat 48 mV/cm for 15 hours; 88 MHz at 48 mV/cm for 15 hours; 71 MHz at 200mV/cm for 30 hours; 73 MHz at 200 mV/cm for 30 hours; 77 MHz at 200mV/cm for 30 hours; 88 MHz at 200 mV/cm for 30 hours. The amount ofdoxycycline in a sample was reduced by more than 33% relative to thecontrol with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

Using cells of Saccharomyces cerevisiae strain IFFI1215 which had beencultured in the presence of a series of 8 EM fields in the order stated:70 MHz at 48 mV/cm for 15 hours; 75 MHz at 48 mV/cm for 15 hours; 82 MHzat 48 mV/cm for 15 hours; 85 MHz at 48 mV/cm for 15 hours; 70 MHz at 200mV/cm for 30 hours; 75 MHz at 200 mV/cm for 30 hours; 82 MHz at 200mV/cm for 30 hours; 85 MHz at 200 mV/cm for 30 hours. The amount oftetracycline in a sample was reduced by more than 26% relative to thecontrol with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

Using cells of Saccharomyces cerevisiae strain IFFI1213 which had beencultured in the presence of a series of 8 EM fields in the order stated:70 MHz at 48 mV/cm for 15 hours; 73 MHz at 48 mV/cm for 15 hours; 80 MHzat 48 mV/cm for 15 hours; 96 MHz at 48 mV/cm for 15 hours; 70 MHz at 200mV/cm for 30 hours; 73 MHz at 200 mV/cm for 30 hours; 80 MHz at 200mV/cm for 30 hours; 96 MHz at 200 mV/cm for 30 hours. The amount ofstreptomycin in a sample was reduced by more than 31% relative to thecontrol with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

Using cells of Saccharomyces cerevisiae strain IFFI1206 which had beencultured in the presence of a series of 8 EM fields in the order stated:71 MHz at 48 mV/cm for 15 hours; 78 MHz at 48 mV/cm for 15 hours; 86 MHzat 48 mV/cm for 15 hours; 98 MHz at 48 mV/cm for 15 hours; 71 MHz at 200mV/cm for 30 hours; 78 MHz at 200 mV/cm for 30 hours; 86 MHz at 200mV/cm for 30 hours; 98 MHz at 200 mV/cm for 30 hours. The amount ofkanamycin in a sample was reduced by more than 25% relative to thecontrol with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

Using cells of Saccharomyces cerevisiae strain IFFI1211 which had beencultured in the presence of a series of 8 EM fields in the order stated:73 MHz at 48 mV/cm for 15 hours; 79 MHz at 48 mV/cm for 15 hours; 88 MHzat 48 mV/cm for 15 hours; 98 MHz at 48 mV/cm for 15 hours; 73 MHz at 200mV/cm for 30 hours; 79 MHz at 200 mV/cm for 30 hours; 88 MHz at 200mV/cm for 30 hours; 98 MHz at 200 mV/cm for 30 hours. The amount oferythromycin in a sample was reduced by more than 27% relative to thecontrol with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

Using cells of Saccharomyces cerevisiae strain IFFI1210 which had beencultured in the presence of a series of 8 EM fields in the order stated:70 MHz at 48 mV/cm for 15 hours; 77 MHz at 48 mV/cm for 15 hours; 84 MHzat 48 mV/cm for 15 hours; 93 MHz at 48 mV/cm for 15 hours; 70 MHz at 200mV/cm for 30 hours; 77 MHz at 200 mV/cm for 30 hours; 84 MHz at 200mV/cm for 30 hours; 93 MHz at 200 mV/cm for 30 hours. The amount ofspiramycin in a sample was reduced by more than 22% relative to thecontrol with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

Using cells of Saccharomyces cerevisiae strain IFFI1260 which had beencultured in the presence of a series of 8 EM fields in the order stated:75 MHz at 48 mV/cm for 15 hours; 78 MHz at 48 mV/cm for 15 hours; 81 MHzat 48 mV/cm for 15 hours; 95 MHz at 48 mV/cm for 15 hours; 75 MHz at 200mV/cm for 30 hours; 78 MHz at 200 mV/cm for 30 hours; 81 MHz at 200mV/cm for 30 hours; 95 MHz at 200 mV/cm for 30 hours. The amount ofbacitracin in a sample was reduced by more than 17% relative to thecontrol with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

5.10. Odor-Reducing Yeast Cell Component

The present invention also provides yeast cells that are capable ofreducing the odor of Sludge. Without being bound by any theory, theinventor believes that the yeast cells of the invention are capable ofreducing the odor of sludge by modifying or decomposing known andunknown compounds in the manure that are malodorous. However, it is notnecessary to demonstrate that such compounds have been decomposed. It issufficient so long as the odor is reduced as determined subjectively bya panel of subjects, after the yeast cells of the invention have beenused.

According to the present invention, yeast cells that are capable ofreducing the odor of organic materials are prepared by culturing thecells in the presence of an electromagnetic field in an appropriateculture medium. The frequency of the electromagnetic field foractivating or enhancing this ability in yeasts can generally be found inthe range of 2160 to 2380 MHz. After sufficient time is given for theyeast cells to grow, the yeast cells can be tested for their ability toreduce the odor of organic materials by methods well known in the art.

The method of the invention for making the odor-reducing yeast cells iscarried out in a liquid medium. The medium contains sources of nutrientsassimilable by the yeast cells. In general, carbohydrates such assugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 0.8%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

Among the inorganic salts which can be incorporated in the culture mediaare the customary salts capable of yielding sodium, calcium, phosphate,sulfate, carbonate, and like ions. Non-limiting examples of nutrientinorganic salts are (NH₄)₂HPO₄, K₂HPO₄, CaCO₃, MgSO₄, NaCl, and CaSO₄.

TABLE 10 Composition for a culture medium for yeasts that reduce odorMedium Composition Quantity Sludge 100 g NaCl 0.2 g MgSO₄.7H₂O 0.2 gCaCO₃.5H₂O 0.5 g CaSO₄.2H₂O 0.2 g K₂HPO₄ 0.5 g Autoclaved water 900 ml

It should be noted that the composition of the media provided in Table10 is not intended to be limiting. Various modifications of the culturemedium may be made by those skilled in the art, in view of practical andeconomic considerations, such as the scale of culture and local supplyof media components.

The process can be initiated by inoculating 100 ml of medium with 1 mlof an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

The EM field(s), which can be applied by any means known in the art, caneach have a frequency in the range of 2160.000 to 2380.000 MHz, andpreferably in the ranges of 2160 to 2250 MHz or 2280 to 2380 MHz. Forexample and without being limited by such examples, each EM field canhave a frequency at about 2160, 2165, 2170, 2175, 2180, 2185, 2190,2195, 2200, 2205, 2210, 2215, 2220, 2225, 2230, 2235, 2240, 2245, 2250,2280, 2285, 2290, 2295, 2300, 2305, 2315, 2320, 2325, 2330, 2335, 2340,2345, 2350, 2355, 2360, 2365, 2370, 2375, or 2380 MHz. The fieldstrength of the EM field(s) is in the range of 0.5 to 320 mV/cm,preferably 30 to 310 mV/cm. If a series of EM fields are applied, the EMfields can each have a different frequency within the stated range, or adifferent field strength within the stated range, or different frequencyand field strength within the stated ranges. In a preferred embodiment,the EM field(s) at the beginning of a series have a lower EM fieldstrength than later EM field(s), such that the yeast cell culture areexposed to EM fields of progressively increasing field strength.Although any practical number of EM fields can be used within a series,it is preferred that the yeast culture be exposed to a total of 2, 3, 4,5, 6, 7, or 8 different EM fields in a series.

Although the yeast cells will become activated even after a few hours ofculturing in the presence of the EM field(s), and the yeast cells can becultured in the presence of the EM field(s) for an extended period oftime (e.g., two or more weeks), it is generally preferred that theactivated yeast cells be allowed to multiply and grow in the presence ofthe EM field or EM fields for a total of about 80-320 hours.

The process of the invention is carried out at temperatures ranging fromabout 23° to 30° C.; however, it is preferable to conduct the process at25° to 28° C. The culturing process may preferably be conducted underconditions in which the concentration of dissolved oxygen is between0.025 to 0.8 mol/m³, preferably 0.4 mol/m³. The oxygen level can becontrolled by any conventional means known to one skilled in the art,including but not limited to stirring and/or bubbling.

At the end of the culturing process, the yeast cells may be recoveredfrom the culture by various methods known in the art, and stored at atemperature below about 0-4° C. The recovered yeast cells may also bedried and stored in powder form.

Any methods known in the art can be used to test the cultured yeastcells for their ability to reduce the odor of organic materials. Theamount of malodorous chemicals such as hydrogen sulfide, ammonia,indole, p-cresol, skatol, and organic acids present in a test sample oforganic material can be determined by any methods known in the art,including but not limited to gas phase chromatography, olfactometry,mass spectrometry, or the use of an odor panel.

To determine the activity of the activated yeast cells towards anmalodorous compound, methods well known in the art, such as HPLC or massspectrometry (e.g., VG micromass), can be used to measure the amounts ofthe malodorous compound in a test sample at various time point and underdifferent incubation conditions. For example, a known amount of amalodorous compound (up to 100 mg per liter) is added to 10 liter of anaqueous extract of manure. Then, 0.1 ml of activated and non-activatedyeasts (at least 10⁷ cells/ml) are added to the 10 liter samplescontaining the antibiotics, and incubated for 24 hours at 28° C. Acontrol is also included which does not contain any yeast cells. After24 hours, the amounts of the malodorous compounds remaining in theextracts are determined and compared.

Accordingly, the odor caused by hydrogen sulfide and other relatedsulfur-containing or sulfhydryl (SH—) containing molecules can bereduced by yeasts cultured in the presence of an EM field that is in therange of 2160.000 to 2250.000. Using cells of Saccharomyces cerevisiaestrain AS2.559 which had been cultured in the presence of a series offour EM fields in the order stated: 2165 MHz at 240 mV/cm for 20 hours;2175 MHz at 240 mV/cm for 20 hours; 2200 MHz at 240 mV/cm for 20 hours;and 2235 MHz at 240 mV/cm for 20 hours, the amount of hydrogen sulfidein a sample was reduced by more than 13% relative to the controlcontaining no yeasts. There was no significant reduction in themalodorous compound in the sample containing non-activated yeasts

The odor caused by ammonia and related NH-containing compounds can bereduced by yeasts cultured in the presence of an EM field that is in therange of 2160.000 to 2250.000. Using cells of Saccharomyces cerevisiaestrain AS2.423 which had been cultured in the presence of a series offour EM fields in the order stated: 2160 MHz at 250 mV/cm for 20 hours;2175 MHz at 250 mV/cm for 20 hours; 2210 MHz at 250 mV/cm for 20 hours;and 2245 MHz at 250 mV/cm for 10 hours, the amount of ammonia in asample was reduced by more than 11% relative to the control containingno yeasts. There was no significant reduction in the malodorous compoundin the sample containing non-activated yeasts.

The odor caused by indole and other related molecules, such as skatol,can be reduced by yeasts cultured in the presence of an EM field that isin the range of 2160.000 to 2250.000. Using cells of Saccharomycescerevisiae strain AS2.612 which had been cultured in the presence of aseries of four EM fields in the order stated: 2165 MHz at 240 mV/cm for40 hours; 2180 MHz at 240 mV/cm for 20 hours; 2200 MHz at 240 mV/cm for40 hours; and 2220 MHz at 240 mV/cm for 20 hours, the amount of indolein a sample was reduced by more than 15% relative to the controlcontaining no yeasts. There was no significant reduction in themalodorous compound in the sample containing non-activated yeasts.

The odor caused by organic acids (such as formic acid, acetic acid,propanoic acid, butyric acid, and other volatile fatty acids) can bereduced by yeasts cultured in the presence of an EM field that is in therange of 2280.000 to 2380.000. Using cells of Saccharomyces cerevisiaestrain AS2.53 which had been cultured in the presence of a series offour EM fields in the order stated: 2315 MHz at 290 mV/cm for 30 hours;2335 MHz at 290 mV/cm for 10 hours; 2355 MHz at 290 mV/cm for 20 hours;and 2375 MHz at 290 mV/cm for 10 hours, the amount of acetic acid in asample was reduced by more than 19% relative to the control containingno yeasts. There was no significant reduction in the malodorous compoundin the sample containing non-activated yeasts.

The odor caused by methylamine, dimethylamine, trimethylamine, and otheraliphatic substituted amines can be reduced by yeasts cultured in thepresence of an EM field that is in the range of 2160.000 to 2250.000.Using cells of Saccharomyces cerevisiae strain AS2.541 which had beencultured in the presence of a series of four EM fields in the orderstated: 2160 MHz at 250 mV/cm for 20 hours; 2190 MHz at 250 mV/cm for 10hours; 2210 MHz at 250 mV/cm for 40 hours; and 2250 MHz at 250 mV/cm for40 hours, the amount of methyl-substituted amides in a sample wasreduced by more than 23% relative to the control containing no yeasts.There was no significant reduction in the malodorous compound in thesample containing non-activated yeasts.

The odor caused by p-cresol and related compounds can be reduced byyeasts cultured in the presence of an EM field that is in the range of2280.000 to 22380.000. Using cells of Saccharomyces cerevisiae strainAS2.163 which had been cultured in the presence of a series of four EMfields in the order stated: 2300, MHz at 98 mV/cm for 20 hours; 2370 MHzat 98 mV/cm for 15 hours; 2300 MHz at 250 mV/cm for 20 hours; and 2370MHz at 250 mV/cm for 30 hours, the amount of p-cresol in a sample wasreduced by more than 23% relative to the control containing no yeasts.There was no significant reduction in the malodorous compound in thesample containing non-activated yeasts.

5.11. Formation of Symbiosis-Like Relationships

In another embodiment of the present invention, yeast cells with thenewly activated or enhanced ability to (1) fix nitrogen, (2) decomposephosphorus-containing minerals or compounds, (3) balance phosphoruscompounds, (4) decompose insoluble potassium-containing minerals orcompounds, and (5) decompose complex carbon compounds as described inSections 5.1-5.5 are combined and cultured so that they form asymbiosis-like relationship whereby they can grow together withoutsubstantially relying on outside supplies of biological availablenitrogen, phosphorus, potassium, and carbon nutrients. The nutrientsneeded for growth are supplied by the respective nutrient-producingyeast strain within the fertilizer composition by convertingbiologically-unavailable nutrients from various sources into availablenutrients. The activity of each of the yeast strains in producing therespective types of nutrient relates in part to the needs of other yeastcells as well as the plants. As a result, soluble,biologically-available nutrients will be converted when needed, therebyavoiding excess losses due to, for example, leaching.

The optional process which can be used to improve the performance of thebiological fertilizer is described as follows. At least four strains ofyeasts prepared according to Sections 5.1-5.5 are mixed and cultured inthe presence of an electromagnetic field in an appropriate liquidmedium. The medium contains nitrogen, phosphorus, potassium, and carbonnutrients in biologically unavailable forms. As non-limiting examples,atmospheric nitrogen is used as the source of nitrogen nutrient, powderof phosphate rock is used as the source of phosphorus nutrient, powderof potassium mica is used as the source of potassium nutrient, andpowdered cellulose is used as the source of complex carbon nutrient.Other forms of insoluble phosphorus and potassium-containing substancesand complex carbon compounds may also be used in place of or incombination with any of the above-identified minerals as sources ofphosphorus, potassium, and carbon nutrients. Among the inorganic saltswhich can be incorporated in the culture media are the customary saltscapable of yielding sodium, calcium, sulfate, carbonate, and like ions.Non-limiting examples of nutrient inorganic salts are CaCO₃, MgSO₄,NaCl, and CaSO₄.

TABLE 11 Composition for a culture medium for formation ofsymbiosis-like relation Medium Composition Quantity NaCl 0.5 gMgSO₄.7H₂O 0.4 g CaCO₃.5H₂O 3.0 g CaSO₄.2H₂O 0.3 g Yeast extract paste0.3 g Potassium mica 1.2 g; Powder of > 200 mesh Rock phosphate 1.2 g;Powder of > 200 mesh Cellulose 5.0 g; Powder of > 200 mesh Autoclavedwater 1000 ml

It should be noted that the composition of the media provided in Table11 is not intended to be limiting. Various modifications of the culturemedium may be made by those skilled in the art, in view of practical andeconomic considerations, such as the scale of culture and local supplyof media components.

The culturing process may preferably be conducted under conditions inwhich the concentration of dissolved oxygen is between 0.025 to 0.8mol/m³, preferably 0.4 mol/m³. The oxygen level can be controlled by anyconventional means known to one skilled in the art, including but notlimited to stirring and/or bubbling. The process of the invention iscarried out at temperatures ranging from about 25° to 30° C.; however,it is preferable to conduct the process at 28° C. The process isinitiated in sterilized medium by inoculating typically about 20 ml ofeach inoculum of the four strains of yeast cells, each at a cell densityof about 10⁸ cell/ml. The optional process can be scaled up or downaccording to needs.

The yeast culture is grown for 12-72 hours, preferably for about 48hours, in the presence of four independent electromagnetic fields. Theelectromagnetic fields, which car be applied by a variety of means, eachhas the following respective frequencies: (1) in the range of about 840to about 916 MHz for nitrogen-fixing; (2) in the range of about 300 toabout 500 MHz for phosphorus-decomposing or phosphorus balancing; (3) inthe range of about 100 to about 300 MHz for potassium-decomposing; and(4) in the range of about 1000 to about 1200 MHz for complexcarbon-decomposing. Generally, the yeast cells are subjected to an EMfield strength in the range from, 5 mV/cm to 160 mV/cm per completecycle. Using an exemplary apparatus as depicted in FIG. 2, the outputamplitude of the EM waves used are in the range of 0-3000 mV, preferably20-1800 mV. The amplitude of each electromagnetic field is repeatedlycycled between 0 mV to 3000 mV, preferably between 20 mV to 1800 mV, insteps of 1 mV at a rate of about two to about ten minutes per completecycle.

5.12. Soil Adaptation

The yeast cells of the invention must also be able to grow and performtheir respective functions in various types of soils. The ability of theyeast cells to survive and grow can be enhanced by adapting the yeastcells of the invention to a particular soil condition.

In another embodiment of the invention, yeast cells prepared accordingto any one of Sections 5.1-5.10 can be cultured separately or in amixture in a solid or semi-solid medium containing soil from one or moresoil sources. This optional process which can be used to improve theperformance of the biological fertilizer described by way of an exampleas follows.

A suspension containing 10 ml of yeasts at a density of 10⁶ cell/ml ismixed with a 1000 cm³ of the soil medium. The process can be scaled upor down according to needs. The mixture of yeast and soil is culturedfor about 48-96 hours, preferably for about 48 hours, in the presence ofan electromagnetic field. The electromagnetic field, which can beapplied by a variety of means, has a frequency that, depending on thefunction of the yeasts, corresponds to one of the frequencies describedin Sections 5.1-5.10. Generally, the yeast cells are subjected to an EMfield strength in the range from 60 mV/cm to 250 mV/cm in this process.

The culture is incubated at temperatures that cycle between about 3° C.to about 48° C. For example, in a typical cycle, the temperature of theculture may start at 35-48° C. and be kept at this temperature for about1-2 hours, then adjusted up to 42-45° C. and kept at this temperaturefor 1-2 hours, then adjusted to 26-30° C. and kept at this temperaturefor about 2-4 hours, and then brought down to 5-10° C. and kept at thistemperature for about 1-2 hours, and then the temperature may be raisedagain to 35-45° C. for another cycle. The cycles are repeated until theprocess is completed. After the last temperature cycle is completed, thetemperature of the culture is lowered to 3-4° C. and kept at thistemperature for about 5-6 hours. After adaptation, the yeast cells maybe isolated and recovered from the medium by conventional methods, suchas filtration. The adapted yeast cells can be stored under 4° C. Anexemplary set-up of the culture process is depicted in FIG. 3.

5.13. Separation or Enrichment of Yeast Cells

Yeast cells that have been adapted to form a symbiosis-like relationshipaccording to Section 5.11 can be separated or enriched in such a waythat each strain of yeast cells keep their acquired or enhancedfunctions. Separation of yeast cells is carried out according to methodsdescribed in U.S. Pat. No. 5,578,486 and Chinese patent publication CN1110317A which are incorporated herein by reference in its entirety. Thesame frequency used for activating the yeast cells may be used duringthe separation process. The separated yeast cells can then be dried, andstored.

5.14. Manufacture of the Biological Fertilizers

In addition to the yeast cell components, sludge and optionallyinorganic materials are also included in the biological fertilizercompositions of the invention. The preparation of manure and suchmaterials as well as the steps involved in the manufacture of thebiological fertilizer compositions are described below.

5.14.1. Preparation of the Organic and Inorganic Substrate Components

Sludge from many varied sources can be used in the biological fertilizercompositions of the present invention. Mixtures of sludge from differentsources can also be used. Organic compounds present in the sludge aredecomposed by the yeasts of the invention. Depending on the source ofthe sludge, in addition to nitrogen, it may contain an useful amount ofphosphorus (e.g., P₂O₅) and potassium (e.g., K₂O). Nutrientconcentrations in sludge, can vary due to its origin, processing if any,methods of storage and moisture content. Methods known in the art can beemployed to determine the nutrient value of each batch of sludge priorto its use in making a biological fertilizer composition.

Inorganic materials, such as but not limited to phosphate rock andpotassium mica, can optionally be included as additional sources ofphosphorus and potassium respectively. Other phosphorous- orpotassium-containing materials and minerals can also be used. Theseinorganic compounds are decomposed by K-decomposing and P-decomposingyeast cells into biologically available potassium and biologicallyavailable phosphorus that can be used by the growing plants as well asthe yeast cells in the fertilizer.

Any inorganic material may be used in combination with sludge in thepresent invention. Alternatively, the inorganic ingredients may beomitted, or substituted by another if it is deemed desirable by theparticular application. For example, phosphate rock can be omitted ifsludge is used which contains sufficient biologically availablephosphorus.

The sludge is preferably dried to a moisture content of ≦5%. Both thedried sludge and optional inorganic substrate components in the presentinvention are ground into suitable forms and sizes before incorporatedinto the fertilizer. Typically, the sludge or inorganic material isconveyed into a crusher where it is broken up into pieces of ≦5 cm indiameter. Any conventional crusher or equivalent machines can be usedfor this purpose. The pieces are then transferred to a grinder by anyconveying means and ground to a powder of ≧150 mesh. Any grinder thatallows fine grinding can be used for this purpose. The powder is thenconveyed to an appropriate storage tank for storage until use with othercomponents of the fertilizer. A schematic illustration of the grindingprocess is shown in FIGS. 4 and 5.

5.14.2. Fermentation Process Using Growth Factor-Producing Yeast

In the present invention, the preparation of GF-producing yeast iscarried out in a fermentation process using as seed the activated yeaststrain as described in Section 5.6. A schematic of the fermentationprocess is illustrated in FIG. 6.

The fermentation medium is prepared according to a ratio of 2.5 litersof water per kilogram of starch. Clean water, preferably water free ofany microorganisms, is used to prepare the fermentation medium. Thefermentation is carried out at a temperature between 20-30° C.,preferably between 25-28° C., in a clean environment and in a spacewhere there are no strong sources of electromagnetic fields, such aspower lines and power generators. Any equipments that contact thefermentation broth, including reactors, pipelines, and stirrers, must bethroughly cleaned before each use. The fermentation process normallylasts about 48-72 hours at 28-30° C. when at least 90% of thefermentation substrate is fermented. Fermentation is preferablyconducted under semi-aerobic conditions or conditions in which theoxygen level is about 20-60% of the maximal soluble oxygenconcentration. The oxygen level can be controlled by any conventionalmeans known to one skilled in the art including but not limited tostirring and/or bubbling. After fermentation, the cell counts shouldreach about 2×10¹⁰ cells/ml. The fermentation broth is kept at atemperature in the range of 15-28° C. and must be used within 24 hours.Alternatively, the GF-producing yeasts an be drained, dried and storedin powder form.

5.14.3. Fermentation Process Using ATP-Producing Yeast

In the present invention, the preparation of ATP-producing yeast iscarried out by a fermentation process using as seed the adapted yeaststrain as described in Section 5.7. A schematic of the fermentationprocess is illustrated in FIG. 6.

The fermentation medium is prepared according to a ratio of 2.5 litersof water per kilogram of starch. Clean water, preferably water free ofany microorganisms, most preferably autoclaved water, is used to preparethe fermentation media. The fermentation is carried out at a temperaturebetween 20-30° C., preferably between 25-28° C., in a clean environmentand in a space where there are no strong sources of electromagneticfields, such as power lines and power generators. Any equipments thatcontact the fermentation broth, including reactors, pipelines, andstirrers, must be throughly cleaned before each use. The fermentationprocess normally lasts about 48-72 hours, depending on the fermentationtemperature. Preferably at the end of the process at least 90% of thefermentation substrate is fermented. Fermentation is preferablyconducted under semi-aerobic conditions or conditions in which theoxygen level is about 20-60% of the maximal soluble oxygenconcentration. The oxygen level can be controlled by any conventionalmeans known to one skilled in the art, including but not limited tostirring and/or bubbling. After fermentation, the cell counts shouldreach about 2×10¹⁰ cells/ml. The fermentation broth is kept at atemperature in the range of 15-28° C. and must be used within 24 hours.Alternatively, the ATP-producing yeasts can be drained, dried and storedin powder form.

5.14.4. Preparation of Mixture of Raw Materials

Sludge and the optional inorganic raw materials are mixed in exemplaryproportions as shown in Table 12. Appropriate amount of organic andinorganic materials prepared according to Section 5.10.1 and starch areconveyed to a mixer. Any conventional mixer, such as but not limited arotary drum mixer, can be used. The mixing tank is rotated constantly sothat powders of sludge and starch are mixed evenly. The mixture is thenconveyed to a storage tank. The procedure for mixing sludge andinorganic substrate material is illustrated in FIG. 7.

TABLE 12 Ratio of raw materials Material Percentage Requirement Powderof dried sludge 58-63% ≧ 150 mesh, water content ≦ 5% Powder ofinorganic materials   20% ≧ 150 mesh, water content ≦ 3% Starch 10-15%regular starch powder, water content ≦ 8%

5.14.5. Preparation of Yeast Mixture

If no inorganic materials is used, the proportion of sludge can beincreased up to 80%. A yeast mixture is prepared in the exemplaryproportions as shown in Table 13. Appropriate amounts of the nine yeaststrains in dried powder form prepared according to Section 5.1-5.10 areconveyed to a mixing tank. The yeasts are allowed to mix for about 10-20minutes. The mixture is then transferred to a storage tank. Anyequipments used for mixing yeasts, including the mixing tank and thestorage tank, must be throughly cleaned, preferably sterilized, beforeeach use. The yeast mixture is stored at a temperature below 20° C. andmust be used within 24 hours. The procedure for mixing yeasts isillustrated in FIG. 8. Alternatively, the mixture of nine yeasts can bedried and stored in powder form.

TABLE 13 Ratio of microorganisms Percentage Yeast Quantity (dry weight)Note Nitrogen-fixing yeast 1.0-2.0 kg 0.1-0.2% Dry yeast powderPhosphorus- 1.0-2.0 kg 0.1-0.2% Dry yeast powder decomposing yeastPotassium- 1.0-2.0 kg 0.1-0.2% Dry yeast powder decomposing yeastCarbon-decomposing 1.0-2.0 kg 0.1-0.2% Dry yeast powder yeastPathogen-suppressing 1.0-2.0 kg 0.1-0.2% Dry yeast powder yeastChemical- 1.0-2.0 kg 0.1-0.2% Dry yeast powder decomposing yeastOdor-reducing yeast 1.0-2.0 kg 0.1-0.2% Dry yeast powder Growth factor-25 L 1% Yeast fermentation producing yeast broth ATP-producing yeast 75L 3% Yeast fermentation broth

5.14.6. Manufacture of Biological Fertilizer

The biological fertilizer of the present invention is produced by mixingthe yeast mixture of Section 5.14.5 and the mixture of the organic andinorganic materials of Section 5.14.1 at a ratio according to Table 14.For example, the yeasts and the sludge, and inorganic materials areconveyed to a granulizer to form granules. The granules of thefertilizer are then dried in a two-stage drying process. During thefirst drying stage, the fertilizer is dried in a first dryer at atemperature not exceeding 65° C. for a period of time not exceeding 10minutes so that yeast cells quickly become dormant. The fertilizer isthen send to a second dryer and dried at a temperature not exceeding 70°C. for a period of time not exceeding 30 minutes to further removewater. After the two stages, the water content should be lower than 5%.It is preferred that the temperatures and drying times be adhered to inboth drying stages so that yeast cells do not lose their vitality andfunctions. The fertilizer is then cooled to room temperature. Thefertilizer may also be screened in a separator so that fertilizergranules of a preferred size are selected. Any separator, such as butnot limited to a turbo separator with adjustable speed and screen sizes,can be used. The fertilizer of the selected size is then sent to a bulkbag filler for packing.

The production process is illustrated in FIGS. 9-11. FIG. 9 is aschematic illustration of the procedure for producing the fertilizerfrom its components. FIG. 10 is a schematic illustration of the dryingprocess. FIG. 11 is a schematic illustration of the cooling and packingprocess.

TABLE 14 Composition of the biological fertilizer (for one metric ton offertilizer) Percentage (dry Quantity weight) Note Mixture of rawmaterials 952-956 kg 95.2-95.4% Dry weight Mixture of yeasts 100 L4.4-4.8% Dry weight

6. EXAMPLE

The following examples demonstrate the manufacture of an exemplarybiological fertilizer composition of the present invention. Theseexamples represent a preferred embodiment of the present invention.

6.1 Biological Fertilizer Composition Comprising Sludge

Saccharomyces cerevisiae strains having accession numbers AS2.628,AS2.631, AS2.982, AS2.413 and AS2.536 are used to prepare the yeast cellcomponents of the biological fertilizer composition. All were depositedin China General Microbiological Culture Collection Center (CGMCC),China Committee for Culture Collection of Microorganisms. Yeast strainAS2.628 is cultured according to the method described in Section 5.1 fornitrogen-fixation; and yeast strain AS2.399 to the method described inSection 5.2 for P-decomposition. Yeast strain AS2.631 is culturedaccording to the method described in Section 5.4 for K-decomposition.Yeast strain AS2.982 is cultured according to the method described inSection 5.5 for C-decomposition. Yeast strain AS2.413 is culturedaccording to the method described in Section 5.6 for production ofgrowth factor. Yeast strain AS2.536 is cultured according to the methoddescribed in Section 5.7 for ATP production. Yeast strain IFFI1301 iscultured according to the method described in Section 5.8 forsuppressing growth of pathogens. Yeast strain IFFI1291 is culturedaccording to the method described in Section 5.9 for degradingundesirable chemicals. Yeast strain IFFI1202 is cultured according tothe method described in Section 5.10 for odor reduction.

Dried sludge in powder form was prepared as described in Section 5.14.5.

The production of growth factor-producing yeast is carried out in afermentation process using as seed the activated yeast strain AS2.413 asdescribed in Section 5.6. A schematic of the fermentation process isillustrated in FIG. 6. The fermentation medium is prepared according toa ratio of 2.5 liters of clean water per kilogram of starch and 10kilograms of starch per metric ton of biological fertilizer. Thefermentation medium is inoculated according to a ratio of 10 ml of seedsolution per liter of medium. The fermentation is carried out at atemperature of 28±1° C. and an oxygen concentration of 0.4 mol/m³ in aclean environment where there were no sources of electromagnetic fields.After about 48 hours of fermentation, the concentration of yeast cellsreached about 2×10¹⁰ cells/ml.

The production of ATP-producing yeast is carried out in a fermentationprocess using as seed the activated yeast strain AS2.536 as described inSection 5.7. A schematic of the fermentation process is illustrated inFIG. 6. The fermentation medium is prepared according to a ratio of 2.5liters of clean water per kilogram of starch and 10 kilograms of starchper metric ton of biological fertilizer. The fermentation medium isinoculated according to a ratio of 10 ml of seed solution per liter ofmedium. The fermentation is carried out at a temperature of 28±1° C. andan oxygen concentration of 0.4 mol/m³ for about 56 hours in a cleanenvironment there were no sources of electromagnetic fields. Afterfermentation, the cell counts reached about 2×10¹⁰ cells/ml.

The mixture of raw materials was prepared according to Table 15 and theprocedure in Section 5.14.5.

TABLE 15 Ratio of raw materials Material Percentage Requirement Driedsludge in powder 80.3% ≧ 150 mesh, water content ≦ form 5% Starch   15%regular starch powder, water content ≦ 8%

The yeast mixture was prepared according to Table 16 and the proceduredescribed in Section 5.14.5.

TABLE 16 Ratio of yeasts (for 1 metric ton of fertilizer) PercentageYeast Quantity (dry weight) Note Nitrogen-fixing yeast 2.0 kg 0.2% Dryyeast AS2.628 powder Phosphorus-decomposing 2.0 kg 0.2% Dry yeast yeastAS2.399 powder Potassium-decomposing yeast 2.0 kg 0.2% Dry yeast AS2.631powder Carbon-decomposing yeast 2.0 kg 0.2% Dry yeast AS2.982 powderPathogen-suppressing yeast 1.0-2.0 kg 0.1-0.2% Dry yeast IFFI1301 powderChemical-degrading yeast 1.0-2.0 kg 0.1-0.2% Dry yeast IFFI1291 powderOdor-reducing yeast 1.0-2.0 kg 0.1-0.2% Dry yeast IFFI1202 powder Growthfactor producing yeast 25 L 1% Yeast AS2.413 fermentation broth ATPproducing yeast AS2.536 75 L 3% Yeast fermentation broth

The biological fertilizer was produced by mixing the yeast mixture,sludge, and any optional inorganic materials at a ratio according toTable 17. The mixed yeasts and sludge were conveyed to a granulizer toform granules. The granules of the fertilizer were then dried in a twostage drying process. During the first drying stage, the fertilizer wasdried in a first dryer at a temperature not exceeding 60±2° C. for aperiod of 5 minutes so that yeast cells quickly became dormant. Thefertilizer was then sent to a second dryer and dried at a temperaturenot exceeding 65±2° C. for a period of 8 minutes to further removewater. The fertilizer was then cool to room temperature. The fertilizerwas then sent to a bulk bag filler for packing.

TABLE 17 Fertilizer composition (for 1 metric ton of fertilizer)Percentage (dry Quantity weight) Note Raw material mixture 949 kg 94.9%Dry weight Yeast mixture 100 L  4.8% Dry weight

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

What is claimed is:
 1. A biological fertilizer composition comprising:(I) sludge; (II) at least one of the following: (a) a first yeast cellcomponent comprising a first plurality of yeast cells characterized byan enhanced ability to fix nitrogen as a result of having been culturedin a first electromagnetic field having a frequency in the range of 840to 916 MHz and a field strength of 10 to 200 mV/cm, as compared to yeastcells not having been so cultured; (b) a second yeast cell componentcomprising a second plurality of yeast cells characterized by anenhanced ability to decompose phosphorous compounds as a result ofhaving been cultured in a second electromagnetic field having afrequency in the range of 300 to 500 MHz and a field strength of 10 to300 mV/cm, as compared to yeast cells not having been so cultured; (c) athird yeast cell component comprising a third plurality of yeast cellscharacterized by an enhanced ability to decompose potassium compounds asa result of having been cultured in a third electromagnetic field havinga frequency in the range of 190 to 285 MHz and a field strength of 10 to200 mV/cm, as compared to yeast cells not having been so cultured; and(III) at least one of the following: (d) a fourth yeast cell componentcomprising a fourth plurality of yeast cells characterized by anenhanced ability to suppress the growth of pathogenic microorganisms asa result of having been cultured in a fourth electromagnetic fieldhaving a frequency in the range of 30 to 50 MHz and a field strength of20 to 200 mV/cm, as compared to yeast cells not having been so cultured;(e) a fifth yeast cell component comprising a fifth plurality of yeastcells characterized by an enhanced ability to degrade antibiotics as aresult of having been cultured in a fifth electromagnetic field having afrequency in the range of 70 to 100 MHz and a field strength in therange of 40 to 250 mV/cm, as compared to yeast cells not having been socultured; and (f) a sixth yeast cell component comprising a sixthplurality of yeast cells characterized by an enhanced ability to reducethe odor of the biological fertilizer composition as a result of havingbeen cultured in a sixth electromagnetic field having a frequency in therange of 2160 to 2250 MHz and 2280 to 2380 MHz and a field strength inthe range of 100 to 300 mV/cm, as compared to yeast cells not havingbeen so cultured.
 2. The biological fertilizer composition of claim 1,further comprising at least one of the following: (g) a seventh yeastcell component comprising a seventh plurality of yeast cellscharacterized by an enhanced ability to convert complex carbon compoundsto simple carbohydrates as a result of having been cultured in a seventhelectromagnetic field having a frequency in the range of 1050 to 1160MHz and a field strength of 10 to 200 mV/cm, as compared to yeast cellsnot having been so cultured; (h) an eighth yeast cell componentcomprising an eighth plurality of yeast cells characterized by anability to overproduce growth factors as a result of having beencultured in an eighth electromagnetic field having a frequency in therange of 1340 to 1440 MHz and a field strength of 20 to 200 mV/cm, ascompared to yeast cells not having been so cultured; and (i) a ninthyeast cell component comprising a ninth plurality of yeast cellscharacterized by an ability to overproduce adenosine triphosphate as aresult of having been cultured in a ninth electromagnetic field having afrequency in the range of 1630 to 1730 MHz and a field strength of 20 to200 mV/cm, as compared to yeast cells not having been so cultured. 3.The biological fertilizer composition of claim 2 wherein each yeast cellcomponent comprises yeast cells of the genus Saccharomyces.
 4. Thebiological fertilizer composition of claim 2 wherein each yeast cellcomponent separately comprises cells of a species of yeast selected fromthe group consisting of Saccharomyces cerevisiae, Saccharomyceschevalieri, Saccharomyces delbrueckii, Saccharomyces exiguus,Saccharomyces fermentati, Saccharomyces logos, Saccharomyces mellis,Saccharomyces microellipsoides, Saccharomyces oviformis, Saccharomycesrosei, Saccharomyces rouxii, Saccharomyces sake, Saccharomyces uvarumBeijer, Saccharomyces willianus, Saccharomyces ludwigii, Saccharomycessinenses, and Saccharomyces carlsbergensis.
 5. The biological fertilizercomposition of claim 2 wherein each yeast cell component separatelycomprises cells of a strain of yeast selected from the group consistingof Saccharomyces cerevisiae Hansen, ACCC2034, ACCC2035, ACCC2036,ACCC2037, ACCC2038, ACCC2039, ACCC2040, ACCC2041, ACCC2042, AS2.1,AS2.4, AS2.11, AS2.14, AS2.16, AS2.56, AS2.69, AS2.70, AS2.93, AS2.98,AS2.101, AS2.109, AS2.110, AS2.112, AS2.139, AS2.173, AS2.174, AS2.182,AS2.196, AS2.242, AS2.336, AS2.346, AS2.369, AS2.374, AS2.375, AS2.379,AS2.380, AS2.382, AS2.390, AS2.393, AS2.395, AS2.396, AS2.397, AS2.398,AS2.399, AS2.400, AS2.406, AS2.408, AS2.409, AS2.413, AS2.414, AS2.415,AS2.416, AS2.422, AS2.423, AS2.430, AS2.431, AS2.432, AS2.451, AS2.452,AS2.453, AS2.458, AS2.460, AS2.463, AS2.467, AS2.486, AS2.501, AS2.502,AS2.503, AS2.504, AS2.516, AS2.535, AS2.536, AS2.558, AS2.560, AS2.561,AS2.562, AS2.576, AS2.593, AS2.594, AS2.614, AS2.620, AS2.628, AS2.631,AS2.666, AS2.982, AS2.1190, AS2.1364, AS2.1396, IFFI 1001, IFFI 1002,IFFI 1005, IFFI 1006, IFFI 1008, IFFI 1009, IFFI 1010, IFFI 1012, IFFI1021, IFFI 1027, IFFI 1037, IFFI 1042, IFFI 1043, IFFI 1045, IFFI 1048,IFFI 1049, IFFI 1050, IFFI 1052, IFFI 1059, IFFI 1060, IFFI 1063, IFFI1202, IFFI 1203, IFFI 1206, IFFI 1209, IFFI 1210, IFFI 1211, IFFI 1212,IFFI 1213, IFFI 1215, IFFI 1220, IFFI 1221, IFFI 1224, IFFI 1247, IFFI1248, IFFI 1251, IFFI 1270, IFFI 1277, IFFI 1287, IFFI 1289, IFFI 1290,IFFI 1291, IFFI 1291, IFFI 1292, IFFI 1293, IFFI 1297, IFFI 1300, IFFI1301, IFFI 1302, IFFI 1307, IFFI 1308, IFFI 1309, IFFI 1310, IFFI 1311,IFFI 1331, IFFI 1335, IFFI 1336, IFFI 1337, IFFI 1338, IFFI 1339, IFFI1340, IFFI 1345, IFFI 1348, IFFI 1396, IFFI 1397, IFFI 1399, IFFI 1411,IFFI 1413; Saccharomyces cerevisiae Hansen Var. ellipsoideus (Hansen)Dekker, ACCC2043, AS2.2, AS2.3, AS2.8, AS2.53, AS2.163, AS2.168,AS2.483, AS2.541, AS2.559, AS2.606, AS2.607, AS2.611, AS2.612;Saccharomyces chevalieri Guillermond, AS2.131, AS2.213; Saccharomycesdelbrueckii, AS2.285; Saccharomyces delbrueckii Lindner var. mongolicusLodder et van Rij, AS2.209, AS2.1157; Saccharomyces exiguus Hansen,AS2.349, AS2.1158; Saccharomyces fermentati (Saito) Ladder et van Rij,AS2.286, AS2.343; Saccharomyces logos van laer et Denamur ex Jorgensen,AS2.156, AS2.327, AS2.335; Saccharomyces mellis Lodder et Kreger VanRij, AS2.195; Saccharomyces microellipsoides Osterwalder, AS2.699;Sacoharomyces oviformis Osterwalder, AS2.100; Saccharomyces rosei(Guilliermond) Ladder et kreger van Rij, AS2.287; Saccharomyces rouxiiBoutroux, AS2.178, AS2.180, AS2.370, AS2.371; Saccharomyces sake Yabe,ACCC2045; Saccharomyces carlsbergensis Hansen, ACCC2032, ACCC2033,AS2.113, AS2.116, AS2.118, AS2.121, AS2.132, AS2.162, AS2.189, AS2.200,AS2.216, AS2.265, AS2.377, AS2.417, AS2.420, AS2.440, AS2.441, AS2.443,AS2.444, AS2.459, AS2.595, AS2.605, AS2.638, AS2.742, AS2.745, AS2.748,AS2.1042; Saccharomyces uvarum Beijer, IFFI 1023, IFFI 1032, IFFI 1036,IFFI 1044, IFFI 1072, IFFI 1205, IFFI 1207; Saccharomyces willianusSaccardo, AS2.5, AS2.7, AS2.119, AS2.152, AS2.293, AS2.381, AS2.392,AS2.434, AS2.614, AS2.1189; Saccharomyces sp., AS2.311; Saccharomycesludwigii Hansen, ACCC2044, AS2.243, AS2.508; and Saccharomyces sinensesYue, AS2.1395.
 6. The biological fertilizer composition of claim 2wherein each yeast cell component comprises cells of Saccharomycescerevisiae.
 7. The biological fertilizer composition of claim 2 furthercomprising an inorganic substrate component.
 8. The biologicalfertilizer composition of claim 7 wherein the inorganic substratecomponent comprises one or more of rock phosphate, apatite, phosphorite,sylvinite, halite, carnalitite, or potassium mica.
 9. The biologicalfertilizer composition of claim 2 which comprises yeast cell components(a) through (f) of claim 1, and yeast cell components (g) thorugh (i) ofclaim
 2. 10. The biological fertilizer composition of claim 2 whereinyeast cell component (a) comprises cells of the yeast Saccharomycescerevisiae AS2.628; yeast cell component (b) comprises cells of theyeast Saccharomyces cerevisiae AS2.399; yeast cell component (c)comprises cells of the yeast Saccharomyces cerevisiae AS2.63 1; yeastcell component (d) comprises cells of one or more of the following yeastSaccharomyces cerevisiae IFFI1037, IFFI1021, IFFI1051, IFFI1331,IFFI1345, or IFFI1211; yeast cell component (e) comprises cells of oneor more of the following yeast Saccharomyces cerevisiae AS2.561,IFFI1063, IFFI1221, IFFI1340, IFFI1215, IFFI1213, IFFI1206, IFFI1211,IFFI1210, or IFFI1260; yeast cell component (f) comprises cells of oneor more of the following yeast Saccharomyces cerevisiae AS2.559,AS2.423, AS2.6 12, AS2.53, AS2.541, or AS2.163; yeast cell component (g)comprises cells of the yeast Saccharomyces cerevisiae AS2.982; yeastcell component (h) comprises cells of the yeast Saccharomyces cerevisiaeAS2.413; and yeast cell component (i) comprises cells of the yeastSaccharomyces cerevisiae AS2.536.
 11. The biological fertilizercomposition of claim 9 wherein yeast cell component (a) comprises cellsof the yeast Saccharomyces cerevisiae AS2.628; yeast cell component (b)comprises cells of the yeast Saccharomyces cerevisiae AS2.399; yeastcell component (c) comprises cells of the yeast Saccharomyces cerevisiaeAS2.63 1; yeast cell component (d) comprises cells of one or more of thefollowing yeast Saccharomyces cerevisiae IFFI1037, IFFI1021, IFFI1051,IFFI1331, IFFI1345, or IFFI1211; yeast cell component (e) comprisescells of one or more of the following yeast Saccharomyces cerevisiaeAS2.561, IFFI1063, IFFI1221, IFFI1340, IFFI1215, IFFI1213, IFFI1206,IFFI1211, IFFI1210, or IFFI1260; yeast cell component (f) comprisescells of one or more of the following yeast Saccharomyces cerevisiaeAS2.559, AS2.423, AS2.61 2, AS2.53, AS2.541, or AS2.163; yeast cellcomponent (g) comprises cells of the yeast Saccharomyces cerevisiaeAS2.982; yeast cell component (h) comprises cells of the yeastSaccharomyces cerevisiae AS2.41 3; and yeast cell component (i)comprises cells of the yeast Saccharomyces cerevisiae AS2.536.
 12. Thebiological fertilizer composition of claim 2, wherein the pluralities ofyeast cells are dried.